Method of producing pharmaceutical compositions comprising immunogenic chikungunya virus chikv-delta5np3

ABSTRACT

The present invention relates to a process for producing an immunogenic live attenuated Chikungunya virus, as well as pharmaceutical compositions comprising the same.

RELATED APPLICATIONS

This Application is a Continuation of U.S. application Ser. No.16/641,012, filed Feb. 21, 2020, entitled “METHOD OF PRODUCINGPHARMACEUTICAL COMPOSITIONS COMPRISING IMMUNOGENIC CHIKUNGUNYA VIRUSCHIKV-DELTA5NP3”, which is a national stage filing under 35 U.S.C. 371of International Patent Application Serial No. PCT/EP2018/075392, filedSep. 19, 2018. The entire contents of these applications areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to a process for producing an immunogenic liveattenuated Chikungunya virus, as well as pharmaceutical compositionscomprising the same.

BACKGROUND OF THE INVENTION

Chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virusfrom the genus Alphavirus, family Togaviridae. Chikungunya virus diseaseis mainly an outbreak disease and is associated with high attack rates.The virus is transmitted to humans via a mosquito vector and causesfever, rash, fatigue and severe polyarthralgia. Infections with CHIKVgenerally resolve spontaneously and are not usually fatal, except inrare cases involving CNS infection, where the death rate is between 10to 30 percent. Particularly at risk for CHIKV CNS disease are infantsunder one year and adults over 65 years, with an infection rate 25-foldand 6-fold higher than the general population, respectively. The rate ofpersistent disabilities in children following CHIKV encephalitis isestimated at between 30 and 45 percent (Gerardin P, et al. Chikungunyavirus-associated encephalitis A cohort study on La Réunion Island,2005-2009 (2016) Neurology 86:1-9). Furthermore, about 30 percent of allChikungunya patients experience arthralgia for months to years afterrecovery. In some cases, neurological, renal, cardiac, respiratory orhepatic complications can occur.

Currently no vaccines or medications are available for the prevention ortreatment of Chikungunya virus disease. Outbreaks in the past haveoccurred mainly in Africa, but the East-Central South African (ECSA)genotype has recently expanded its geographical range, resulting inoutbreaks in India, Asia, and even temperate Europe (Weaver, S., Arrivalof Chikungunya Virus in the New World: Prospects for Spread and Impacton Public Health (2014) PLOS Neglected Tropical Diseases 8(6): e2921).Although CHIKV has been repeatedly imported into the Americas since1995, autochthonous transmission had not been reported until 2013 in theCaribbean. By 2015, the epidemic had spread to the mainland and causedupwards of one million suspected cases in 27 countries in the Americas(Pan-American Health Organization (2015) Number of Cumulative Cases ofChikungunya Fever in the Americas). Further epidemics may been aided inpart by the spread of the CHIKV mosquito vector into non-endemicregions, as well as the ability of CHIKV to adapt to local mosquitospecies (Vega-Rua A, et al., Chikungunya Virus Transmission Potential byLocal Aedes Mosquitoes in the Americas and Europe (2015) PLOS NeglectedTropical Diseases DOI:10.1371/joumal.pntd.0003780). The high rate ofcontagion of Chikungunya virus, its geographical spread, and itspotential for long-lasting complications underscore the need fordeveloping preventative measures, such as vaccines.

Vaccines against Chikungunya virus may comprise live attenuated CHIKVparticles; i.e., live CHIKV particles which have been altered to reducevirulence, but still maintain immunogenicity. One example of anattenuated CHIKV contains a deletion mutation in the non-structuralprotein 3 (CHIKV-Δ5nsP3; see FIG. 2). CHIKV-Δ5nsP3 has been shown toconfer protective immunity in mice (Hallengärd D, et al. Novelattenuated Chikungunya vaccine candidates elicit protective immunity inC57Bl/6 mice (2014) J. Virol. 88:2858-2866) and non-human primates(Roques P, et al. Attenuated and vectored vaccines protect non-humanprimates against Chikungunya virus (2017) J. Clin. Invest. Insight2(6):e83527). These preliminary in vivo studies in mice and non-humanprimates were done with CHIKV-Δ5nsP3 virus produced on BHK-21 cells—acell type generally not favored for the production of human vaccines. Itwould be necessary, therefore, as disclosed herein, to adaptCHIKV-Δ5nsP3 virus production to a more suitable cell culture platform.Such adaptation is not a trivial process; for example, it is known inthe art that the adaptation of viruses to a particular host cell canlead to mutations that change the surface charge of the virus particles.Such acquired mutations can serve to attenuate viruses; in fact, serialpassaging has been used to develop such attenuated virus particles thatare used in many vaccines against viruses. With regard to CHIKV, it hasbeen shown that repeated in vitro passaging of virulent wild-typeChikungunya virus can lead to certain point mutations, resulting inpartial or complete attenuation of the virus (Gardner C L, et al.,Deliberate Attenuation of Chikungunya Virus by Adaptation to HeparanSulfate-Dependent Infectivity: A Model for Rational Arboviral VaccineDesign (2014) PLOS Neglected Tropical Diseases 8(2): e2719).

It has now surprisingly been found that certain point mutationsresulting in loss of immunogenicity can occur early during the cellsubstrate adaptation process of the attenuated CHIKV-Δ5nsP3, makingcontrol or reduction of said point mutations an essential considerationfor the production of a successful vaccine candidate. Thus, the currentinvention provides a process with well-defined parameters for thepropagation of the CHIKV-Δ5nsP3 vaccine candidate, allowing productionof highly immunogenic virus particles, while simultaneously achievinghigh production titers in cell culture suitable for industrialapplication.

SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition comprisinga sufficient amount of immunogenic Chikungunya virus to elicit aneutralizing immune response in a subject; i.e., an immune response thatis protective against infection with and/or disease caused byChikungunya virus. In particular, the invention provides apharmaceutical composition comprising live attenuated CHIKV-Δ5nsP3particles wherein the percentage of said viral particles withimmunogenicity-reducing mutations, particularly immunogenicity-reducingmutations in the E2 protein, are minimized. The disclosure furtherprovides a process for producing a pharmaceutical composition comprisinga live attenuated CHIKV-Δ5nsP3, wherein the process minimizes thepresence of immunogenicity-reducing mutations in the viral genome,particularly mutations at E168 of viral E2 protein and/or other E2residues and/or residues in other structural or non-structural CHIKVproteins. The current disclosure further provides pharmaceuticalcompositions comprising an immunogenic live attenuated Chikungunya virusobtainable by the process of the invention.

Efforts to develop a vaccine against Chikungunya virus are currentlyunderway. One of the most advanced vaccine candidates provides achimeric construct in a measles virus platform (seehttp://www.themisbio.com/#/news). The vaccine, currently in Phase 2trials, is delivered in two doses(https://clinicaltrials.gov/ct2/show/NCT02861586?term=themis&recrs=a&rank=2).A one-shot vaccine would represent a distinct advantage in the field.

Accordingly, in one embodiment, it is an object of the current inventionto provide a stable, well-defined, safe and effective pharmaceuticalcomposition such as, e.g. a vaccine, against Chikungunya virus,preferably an improved vaccine conferring protection with only onevaccination; i.e., a so called “one-shot” vaccine at industrial scaleusing common cell substrates, to provide processes to generate such astable, well-defined, safe and effective vaccine and methods and usesfor said stable, well-defined safe and effective vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. TheFigures are illustrative only and are not required for enablement of thedisclosure. For purposes of clarity, not every component may be labeledin every drawing. In the drawings:

FIG. 1 Map of pMX plasmid used for full assembly of the CHIKV-Δ5nsP3genome from synthesized fragments. For the full assembly of theCHIKV-Δ5nsP3 sequence, the pMX plasmid with an Ampicillin resistancecassette (pMA) was used. The pMX vector series is based on pUC-likecloning vectors, but leaves out unnecessary promoters (biosafety level1).

FIG. 2 Schematic illustration of the CHIKV-Δ5nsP3 genome structure. TheChikungunya virus genome encodes two polyproteins: non-structuralproteins 1-4 (nsP1-4) and structural proteins (C, E3, E2, 6K, E1).Compared with the wild-type genomic sequence, the CHIKV-Δ5nsP3 sequencecontains a 183-bp deletion in the 3′ part of the sequence encoding nsP3(amino acids 1656 to 1717 in the nsP1-4 polyprotein), which results in a60 amino acid deletion in the nsP3 replicase protein (indicated byΔ60aa). SP, subgenomic promoter; UTR, untranslated region. (Figureadapted from Hallengärd D, et al., 2014, supra.)

FIGS. 3A and 3B Cloning strategy for assembly of the CHIKV-Δ5nsP3 genomein pMA. (FIG. 3A) Design schematic of synthesized polynucleotidefragments covering the full CHIKV-Δ5nsP3 genome. (FIG. 3B) Cloningstrategy for assembly of CHIKV-Δ5nsP3 genome in pMA plasmid. 1. Cloningof CHIKV-Δ5nsP3 fragment 2 into pMA containing fragment 1 (pMAfragment 1) via EcoRI and PacI. 2. Assembly of fragment 4 and fragment 3via ClaI and PacI. 3. Preparation of fragment 5 digested with XhoI andPacI in pMA for final full assembly. 4. Full assembly of CHIKV-Δ5nsP3genome in pMA by fusion of AgeI/XhoI-digested fragments 3 and 4 andXhoI/PacI-digested fragment 5 with AgeI/PacI-linearized pMA fragments 1and 2. Correct CHIKV-Δ5nsP3 genome assembly was verified via Sangersequencing.

FIGS. 4A-4C Yield, plaque size and immunogenicity of CHIKV-Δ5nsP3following passaging in Vero cells. Virus was passaged in three parallelreplicates (A, B and C) starting from a common P0 (rescue) as detailedin Table 2. (FIG. 4A) Virus titers 24 h after infection of Vero cellsfrom passage 0 to passage 16. The average titer of the three replicates(A, B and C) is shown. (FIG. 4B) Relative titers of P0, P5 and P15CHIKV-Δ5nsP3 as assessed by plaque assay. Vero cells were seeded at adensity of 4×10⁵ cells per well in 6-well plates in MEM supplementedwith 5% FBS, 2 mM L-Glutamine and 1% Antibiotic-Antimycotic (Anti-Anti)and were incubated overnight at 35° C. and 5% CO₂. On the next day,culture supernatant was removed from Vero cells and serial dilutions ofCHIKV-Δ5nsP3 were added onto the cells. Following incubation for 1 hourat 35° C./5% CO₂, a methylcellulose overlay with a final concentrationof 2% was added and cells were further incubated 3 days at 35° C./5%CO₂. Finally, plaques were counted after crystal violet staining (0.5%crystal violet in 5% formaldehyde) to assess virus titer (pfu/ml) andplaque morphologies. (FIG. 4C) Immunogenicity of P0, P5B, P8B and P15CCHIKV-Δ5nsP3 as assessed by neutralization of CHIKV-Δ5nsP3 (P0) in PRNTon Vero cells. Vero cells were seeded in 12-well plates at a density of3×10⁵ and incubated overnight at 35° C./5% CO₂. Groups of five C57Bl/6mice were immunized once subcutaneously with a dose of 10⁵ TCID₅₀ of therespective CHIKV-Δ5nsP3 passages. CHIKV-Δ5nsP3 at P0 (virus rescue),also at 10⁵ TCID₅₀, was used as a positive control. Day 21 serum poolsat 4-fold serial dilutions ranging from 1:20 to 1:327,680 were mixedwith 560 pfu/ml CHIKV-Δ5nsP3 (at P0) and incubated for one hour. TheCHIKV-Δ5nsP3/neutralization mixes were then added to the Vero cells andthe plates were incubated for 2 hours at 35° C./5% CO₂. This step wasfollowed by a 2% methylcellulose overlay and plates were incubated for˜60 hours at 35° C./5% CO₂. After removal of the overlay, cells werestained with crystal violet/5% formaldehyde and plaques were counted.

FIGS. 5A-5B Effect of controlled MOI (0.01) during passaging ofCHIKV-Δ5nsP3 on immunogenicity. (FIG. 5A) Schematic illustration ofCHIKV-Δ5nsP3 passaging on Vero cells under uncontrolled and controlledconditions. CHIKV-Δ5nsP3 P0 was passaged on Vero cells to P3B underuncontrolled conditions with varying MOI (as outlined in Table 2;Replicate B). Using P3B as a starting material, a controlled infectionprocess was carried out with all subsequent infections at the definedMOI of 0.01 to generate one P4 passage, two P5 passages and one P6passage for analysis of immunogenicity in mice. (FIG. 5B) Immunogenicityof P0 (∘), P2B (□), P5 #1 (●), P5 #2 (♦), P6 (▪) and P15 (Δ)CHIKV-Δ5nsP3 as assessed by neutralization of CHIKV-Δ5nsP3 (P2) in PRNTon Vero cells. Groups of ten C57Bl/6 mice were subcutaneously immunizedwith a single dose of the respective CHIKV-Δ5nsP3 preparations at anintended dose of 10⁵ TCID₅₀ and at day 21 following immunization, pooledsera were assessed for CHIKV-Δ5nsP3 (560 pfu/ml) neutralization capacityat 4-fold serial dilutions ranging from 1:20 to 1:327,680 as describedfor FIGS. 4A-4C.

FIGS. 6A-6D Immunogenicity and observed genomic heterogeneities ofsingle plaque isolates of CHIKV-Δ5nsP3 from P5B and P8B. TwoCHIKV-Δ5nsP3 virus rescue harvests (4399gr1 P0 (#1) and 4415gr1 P0 (#2))and a P15 CHIKV-Δ5nsP3 harvest (P15C-DS) served as immunogenic andnon-immunogenic controls, respectively. (FIG. 6A) Immunogenicity ofCHIKV-Δ5nsP3 single plaque isolates P5B-11, P5B-03 and P8B-05 asassessed by neutralization of CHIKV-Δ5nsP3 (P2) in PRNT on Vero cells.Groups of ten C57Bl/6 mice were subcutaneously immunized with a singledose of the respective CHIKV-Δ5nsP3 isolates at the indicated TCID₅₀doses and at day 19 following immunization, pooled sera were assessedfor CHIKV-Δ5nsP3 (560 pfu/ml) neutralization capacity at 4-fold serialdilutions ranging from 1:20 to 1:327.680 as described for FIGS. 4A-4C.(FIG. 6B) Schematic genomes of CHIKV-Δ5nsP3 single plaque isolatesP5B-11, P5B-03 and P8B-05 derived from Sanger sequencing covering thefull CHIKV-Δ5nsP3 genome with identified point mutations indicated.(FIG. 6C) Immunogenicity of further CHIKV-Δ5nsP3 single plaque isolatesP5B-02, P5B-04, P5B-07 and P8B-01 was assessed as in (A). (FIG. 6D)Schematic genomes of CHIKV-Δ5nsP3 single plaque isolates P5B-02, P8B-01,P5B-04 and P5B-07 derived from Sanger sequencing covering the fullCHIKV-Δ5nsP3 genome with identified point mutations indicated.

FIG. 7 Chromatograms from Sanger sequencing of CHIKV-Δ5nsP3 at passage 0(P0) and four independently generated CHIKV-Δ5nsP3 samples at passage 3(P3 Examples 1-4), revealing E168K and E247K heterogeneities in the E2protein arising by passage 3. The four independently generatedCHIKV-Δ5nsP3 samples at P3 (P3 Examples 1-4) were all derived from thesame MVSB (P1). The regions shown cover genomic regions encoding aminoacids 168 and 247 of the E2 protein and were sequenced with primer pairs16 and 17 as indicated. (For primer sequences, see Table 1). Forcomparison, sequencing chromatograms of CHIKV-Δ5nsP3 at passage 0 (P0)are shown. Sites of sequence heterogeneities are indicated by boxes.

FIGS. 8A-8C Next generation sequencing (NGS) of a Master Virus Seed Bank(P1) and two independently generated passage three (P3) CHIKV-Δ5nsP3preparations: (FIG. 8A) P1-MVSB; (FIG. 8B) P3-Example 3; (FIG. 8C)P3-Example 4. A background level of genomic heterogeneities is set at15% as indicated by the dotted line. More frequent heterogeneitiesarising by passage 3 included point mutations at genomic nucleic acidpositions 8882 and 9119, which correspond to E168K and E247K mutations,respectively, in the E2 protein as indicated in (FIG. 8B) and (FIG. 8C).

FIGS. 9A-9C Location of amino acid positions with frequently observedheterogeneities (G55R, G82R, E168K and E247K) within the E2 glycoproteinmarked on equivalent positions on a cryo-EM structure of CHIKV VLPs ofstrain Senegal 37997 (PDB 3J2W; Sun S, et al., Structural analyses atpseudo atomic resolution of Chikungunya virus and antibodies showmechanisms of neutralization (2013) eLife 2:e00435. DOI:10.7554/eLife.00435). G55R is indicated by a square, G82R is indicatedby a circle, E168K is indicated by a triangle and E247K is indicated bya star. The figures show the CHIKV proteins of the unit-cell as theywould be assembled in the viral membrane, composed of four copies ofE1/E2 dimers with the transmembrane portion (TM) indicated by brackets,and the capsid protein (C) at the inner (bottom) side of the membrane.Three E1/E2/capsid-assemblies compose the trimeric q3-spike near the5-fold axis and the fourth assembly is one of three elements of thei3-spike at the 3-fold axis, all in surface representation and viewedfrom three different angles: (FIG. 9A) CHIKV unit-cell side view;viewpoint above membrane plane. (FIG. 9B) CHIKV unit-cell side view;viewpoint below membrane plane (FIG. 9C) CHIKV unit-cell, top view.

FIG. 10 Groups for subcutaneous immunization of C57Bl/6 mice with viruspreparations of different P3:P5B-07 (E168K) ratios to determine thethreshold at which the presence of the E168K point mutation results inloss of CHIKV-Δ5nsP3 immunogenicity at a dose of approximately 3×10⁴TCID₅₀. The formulation ratios of P3 and P5B-07 (E168K mutant) forgroups 1, 3 and 5 were 1:0.1, 1:1 and 1:10, respectively, with anintended dose of 3×10⁴ TCID₅₀. Additionally, group 6 and group 7represent P3 alone and P5B-07 (E168K) alone, respectively. The referencesequence (wild-type) in the heterogenic position (corresponding tonucleotide 8882) was G and depending on the ratio of either 1:0.1, 1:1or 1:10, a shift towards the nucleotide A, i.e., G>A to G=A or G<A, wasin accordance with sequencing chromatograms. The P3 viral populationdisplayed ˜20% E168K mutants. The P5B-07 (E168K) viral population didnot show heterogeneity (i.e., ˜100% contained the E2 protein E168Kmutation).

FIGS. 11A-11D Effect on immunogenicity of different P3 to E168K-mutantCHIKV-Δ5nsP3 ratios assessed by VRP-based neutralization assay. Singlemouse sera were generated by subcutaneous immunization of C57Bl/6 micewith an intended dose of 3×10⁴ TCID₅₀ of CHIKV-Δ5nsP3 P3:E168K at ratios1:0.1, 1:1 and 1:10 as outlined in FIG. 10. Day 21 single sera wereanalyzed for neutralization of LR-CHIKV virus replicon particles (VRPs)using a BHK-21 cell-based Luciferase assay. VRPs resemble areplication-deficient wild-type CHIKV displaying capsid and envelopeproteins of the LR2006-OPY1 wild-type virus. Briefly, 2×10⁴ BHK-21 cellswere seeded in 96-well plates and incubated overnight at 35° C./5% CO₂.CHIKV-Δ5nsP3 VRPs were incubated with serial dilutions (starting at 1:20to 1:312.500) of mouse sera for 1 hour at 35° C. CHIKV VRP/serum mixeswere added to BHK-21 cells at an MOI of 5 and incubated for 1 hour at35° C./5% CO₂. Cells were washed and fresh medium was added. Luciferaseactivity was measured in the supernatant 24 hours post-infection usingthe Renilla Luciferase Assay System (Promega). P-MVSB positive control(∘), P5B-07 (E168K) negative control (□) and individual mouse seraimmunized with respective P3:E168K ratio (filled circles). (FIG. 11A)Immunogenicity of CHIKV-Δ5nsP3 P3:E168K at a ratio of 1:0.1 (Group 1).(FIG. 11B) Immunogenicity of CHIKV-Δ5nsP3 P3:E168K at a ratio of 1:1(Group 3). (FIG. 11C) Immunogenicity of CHIKV-Δ5nsP3 P3:E168K at a ratioof 1:10 (Group 5). (FIG. 11D) Summary of immunogenicity results shown inA, B, and C: strong immunogenic (++ positive), low immunogenic (+lowpositive) and negative (−) in comparison to P3.

FIG. 12 Vero cell culture train from thawing to infection withCHIKV-Δ5nsP3 in 850 cm² roller bottles. P: Passage. M: Million, T75:T-Flask 75 cm², T175: T-Flask 175 cm², RB850: Roller Bottle CellBIND 850cm².

FIGS. 13A-13C Effect on virus titer of cell growth temperature,multiplicity of infection and post-seeding infection time duringproduction of CHIKV-Δ5nsP3 in Vero cells. Vero cells were expanded at35° C. in MEM, 2 mM glutamine and 10% FBS and seeded in 850 cm² rollerbottles for cell infection. For virus production, different temperatures(37° C. 35° C., 28° C.). MOIs (0.1; 0.01; 0.001 TCID₅₀/cell) and timesof cell infection post Vero cell seeding in days (D2, D4, D5) weretested in culture medium deprived of FBS. Shown are viral productivitiesmeasured on Vero cells according to the Reed & Muench method andexpressed in TCID₅₀/mL. (FIG. 13A) 37° C.; (FIG. 13B) 35° C.; (FIG. 13C)28° C.

FIG. 14 Total CHIKV-Δ5nsP3 virus productivity from each of theconditions described in FIG. 13.

FIGS. 15A-15B Maximum CHIKV-Δ5nsP3 virus titers: Response surfacequadratic model. (FIG. 15A) ANOVA analysis of the model. Grey line:significant model term (Prob(F)<0.05; values greater than 0.1 indicatethe model term is not significant). (FIG. 15B) Contour Plot of themodel.

FIGS. 16A-16B CHIKV-Δ5nsP3 virus stability: Response surface quadraticmodel. (FIG. 16A) ANOVA analysis of the model. Grey line: significantmodel term (Prob(F)<0.05: values greater than 0.1 indicate the modelterm is not significant). (FIG. 16B) Contour Plot of the model.

FIGS. 17A-17B Heterogeneity of the CHIKV-Δ5nsP3 E2 viral protein atgenomic nucleic acid positions 8882, 9112 and 9649, corresponding to E2amino acids 168, 247 and 423, respectively: Model analysis from Day 2CHIKV-Δ5nsP3 sample harvests. (FIG. 17A) ANOVA analysis of the model.Grey line: significant model term (Prob(F)<0.05). (FIG. 17B) ContourPlot of the model.

FIGS. 18A-18B Heterogeneity of the CHIKV-Δ5nsP3 E2 viral protein atgenomic nucleic acid positions 8882, 9112 and 9649; corresponding to E2amino acids 168, 247 and 423, respectively: Model analysis from Day 2and Day 5 CHIKV-Δ5nsP3 sample harvests (28° C.). (FIG. 18A) ANOVAanalysis of the model. Grey line: significant model term (Prob(F)<0.05).(FIG. 18B) Contour Plot of the model.

DETAILED DESCRIPTION OF THE INVENTION

During the course of industrialization of the CHIKV-Δ5nsP3 attenuatedvirus vaccine candidate, it was observed that passaging of the virus onVero cells resulted in higher virus titers with increasing passages;however, a concomitant increase in sequence heterogeneity of theCHIKV-Δ5nsP3 viral genome was also observed. Certain point mutationsarising during passaging on Vero cells were found to be reproduciblefrom batch to batch and appeared already in early passages on the newcell substrate. It was surprisingly observed that some of thesemutations correlated with a significant loss of or decrease inneutralizing immunogenicity conferred by the CHIKV-Δ5nsP3 virus. Otherreproducible mutations did not reduce immunogenicity and/or acted as“rescuing” mutations for the immunogenicity-reducing mutations. Acorrelation between a low multiplicity of infection (“MOI”) andgeneration of increased sequence heterogeneity in CHIKV-Δ5nsP3 wasidentified; however, because of the need to have a single source ofvirus over years of manufacturing, high MOIs are generally not feasiblefor industrial use. It was therefore not clear at the outset whetherculturing conditions allowing the generation of immunogenic CHIKV-Δ5nsP3particles with a production yield sufficient for reproducible andreliable manufacturing could be found (problem of the invention).

Provided herein are methods to control and minimize the herein observedimmunogenicity-reducing mutations while still enabling high productionyields. Also provided herein are pharmaceutical compositions comprisingan effective amount of an immunogenic Chikungunya virus with a residualamount of a non-immunogenic variant of Chikungunya virus. In a preferredembodiment, the pharmaceutical composition is produced using a low MOIsuch as an MOI of less than 0.1, e.g. 0.01 or 0.001, but produced undersuch controlled conditions (e.g., reduced passage numbers followingrescue, optimized temperature and host cell confluency) to minimizeamounts of non-immunogenic variant(s) of Chikungunya virus as describedherein. In some embodiments, the virus particle is a live virus, achimeric virus, an attenuated live virus, a modified live virus, or arecombinant live virus. In one embodiment, the virus particles of theinvention may be optionally inactivated. In some embodiments, the virusparticle is an attenuated form of the virus particle. For example, thevirus may have reduced infectivity, virulence, and/or replication in ahost, as compared to a wild-type virus. In some embodiments, the virusis a mutated or modified virus, for example the nucleic acid of thevirus may contain at least one mutation relative to the wild-type virus,such as a substitution or deletion. In some embodiments, the virus is arecombinant live virus, meaning a virus that is generated recombinantlyand may contain nucleic acid sequences from different sources. In someaspects, the wild-type Chikungunya virus is inactivated. In a preferredembodiment, the virus is inactivated with formaldehyde.

In one embodiment, the immunogenic Chikungunya virus is a liveattenuated virus. In a preferred embodiment, the live attenuatedChikungunya virus is the protective CHIKV-Δ5nsP3 as described byHallengärd D, et al. (supra), referred to herein as CHIKV-Δ5nsP3 anddefined by the nucleic acid sequence of SEQ ID NO: 1. Briefly, thewild-type CHIKV genome carries a positive-sense single-stranded RNAgenome of 11 kb containing two open reading frames encodingnonstructural proteins (nsP1 to nsP4) and structural proteins (C, E3,E2, 6K, and E1), respectively. An attenuated CHIK virus. CHIKV-Δ5nsP3,based on the La Reunion CHIKV strain LR2006-OPY1, was constructed bysubstituting amino acid residues 1656 to 1717 of the P1234 polyproteinwith a small linker (AA sequence AYRAAAG) in the hypervariable region ofthe nsP3 protein (see FIG. 2). CHIKV-Δ5nsP3 has been shown to beinfectious, highly immunogenic and protective against challenge withwild-type CHIKV (Hallengärd D, et al., supra and Hallengärd D. et al.,Prime-Boost Immunization Strategies against Chikungunya Virus (2014) J.Virology, 88(22):13333-13343, and Roques P, et al. 2017, supra). In oneembodiment, the live attenuated Chikungunya virus may be a variant ofthe CHIKV-Δ5nsP3 attenuated mutant virus. In a preferred embodiment, thelive attenuated Chikungunya virus as provided herein comprises theCHIKV-Δ5nsP3 as encoded by the nucleic acid sequence defined by SEQ IDNO: 1. As used herein, the term “CHIKV-Δ5nsP3” is used interchangeablywith “CHIKV-Δ5nsP3 virus”. “CHIKV-Δ5nsP3 particle”. “CHIKV-Δ5nsP3 virusparticle” or plural versions thereof.

Provided herein is a pharmaceutical composition comprising an effectiveamount of a CHIKV-Δ5nsP3. In one aspect, an effective amount of animmunogenic CHIKV-Δ5nsP3 virus is defined as an amount sufficient toelicit neutralizing antibodies to Chikungunya virus. In a furtheraspect, an effective amount of an immunogenic CHIKV-Δ5nsP3 virus isdefined as an amount to elicit an immune response in a vaccinatedsubject which confers protective immunity against Chikungunya virusinfection. In a preferred aspect, an effective amount of CHIKV-Δ5nsP3 isdefined as at least 10², at least 10³, at least 10⁴, at least 10⁵, atleast 10⁶, preferably at least 10³ immunogenic CHIKV-Δ5nsP3 particles.In one aspect, immunogenic CHIKV-Δ5nsP3 particles are defined asCHIKV-Δ5nsP3 particles which express an E2 structural protein as definedby the polypeptide sequence of SEQ ID NO: 2. In one aspect, the E2structural protein contains one or more point mutations that do notaffect the immunogenicity of the virus, i.e., are not immunogenicityreducing. In one embodiment, the point mutations that do not affect theimmunogenicity of the virus may be at amino acids 232 and/or 247 of theE2 protein, such as H232Y and/or E247K. In one embodiment, the E2structural protein of the CHIKV-Δ5nsP3 contains no more than about tenpoint mutations. In one embodiment, the E2 structural protein of theCHIKV-Δ5nsP3 contains no more than 9, 8, 7, 6, 5 or 4 point mutations.In a preferred embodiment, the E2 structural protein of the CHIKV-Δ5nsP3contains three or less point mutations, most preferably only one or twopoint mutations.

As defined herein, an immunogenic CHIKV-Δ5nsP3 is a CHIKV-Δ5nsP3 whichis capable of stimulating an effective immune response in vivo whendelivered e.g. at a dose of about 3×10⁴ TCID₅₀, i.e., an immune responsein which neutralizing antibodies are produced which are sufficient forreducing or preventing signs or symptoms of Chikungunya virus disease.In a preferred embodiment, the immunogenic CHIKV-Δ5nsP3 as definedherein is a CHIKV-Δ5nsP3 which expresses an E2 structural proteinaccording to the amino acid sequence provided by SEQ ID NO: 2. In afurther preferred embodiment, the immunogenic CHIKV-Δ5nsP3 as definedherein is defined by the polynucleotide sequence according to SEQ IDNO: 1. As an alternative or additional definition, the immunogenicCHIKV-Δ5nsP3 of the current invention stimulates the production ofantibodies with neutralizing capacity in an immunized subject, i.e.,neutralization of Chikungunya virus in an in vitro assay of at least50%, preferably at least 60%, preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, most preferably atleast 95% at a 1:80 or higher serum dilution.

As defined herein, a non-immunogenic CHIKV-Δ5nsP3 is a CHIKV-Δ5nsP3which elicits levels of neutralizing antibodies in a vaccinated subjectinadequate to prevent signs or symptoms of Chikungunya virus disease. Ina preferred embodiment, a non-immunogenic CHIKV-Δ5nsP3 is a CHIKV-Δ5nsP3which expresses an E2 structural protein with at least one amino acidsubstitution, especially amino acid substitutions in the E2 structuralprotein, especially E168K and/or G55R substitutions, particularly anE168K substitution. A non-immunogenic CHIKV-Δ5nsP3 is further defined aseliciting antibodies in an immunized subject which show poor capacity toneutralize infection of cells with Chikungunya virus (wild-type orattenuated) in an in vitro assay. In particular, a non-immunogenicCHIKV-Δ5nsP3 is defined as eliciting levels of neutralizing antibodiesin an immunized subject which provide less than 60%, less than 50%, lessthan 40%, less than 30%, less than 20%, especially less than 10%,neutralization of Chikungunya virus in an in vitro neutralization assayat a 1:80 or higher serum dilution.

In a further aspect, the effective amount of CHIKV-Δ5nsP3 is defined asan amount sufficient to elicit neutralizing antibodies against wild-typeChikungunya virus. In one aspect, the pharmaceutical composition is atwo-shot pharmaceutical composition. In a preferred aspect, thepharmaceutical composition is a one-shot pharmaceutical composition. Ina preferred aspect, the pharmaceutical composition comprises at least10², at least 10³, at least 10⁴, at least 10⁵, at least 10⁶, preferablybetween about 10³ to 10⁴ total CHIKV-Δ5nsP3 viral particles, especiallyabout 10³ or 10⁴ CHIKV-Δ5nsP3 comprised in a total pool of particleswith and without point mutations, especially immunogenicity-reducingpoint mutations. In a preferred aspect, the pharmaceutical compositioncomprises a detectable amount of non-immunogenic CHIKV-Δ5nsP3 as definedherein; preferably a non-immunogenic CHIKV-Δ5nsP3 with at least onepoint mutation compared with the wild-type E2 protein as defined by SEQID NO: 2.

In a preferred embodiment, the pharmaceutical composition comprisesCHIKV-Δ5nsP3 and comprises an increased amount of a non-immunogenicvariant(s) of CHIKV-Δ5nsP3, e.g. compared to a vaccine compositioncomprising CHIKV-Δ5nsP3 produced in BHK-21 cells as used in mousestudies described in Hallengärd D, et al. 2014, supra, but stillcomprises sufficient immunogenic particles of CHIKV-Δ5nsP3 to produceprotective immunity in a vaccinated subject. For instance, thepharmaceutical composition may comprise (i) CHIKV-Δ5nsP3 which expressesan E2 structural protein as defined by the polypeptide sequence of SEQID NO: 2 in an amount sufficient to produce protective immunity in avaccinated subject; (ii) an increased amount of CHIKV-Δ5nsP3 having atleast one mutation in said E2 structural protein, e.g. compared to avaccine composition comprising CHIKV-Δ5nsP3 produced in BHK-21 as usedin mouse studies described in Hallengärd D, et al. 2014, supra; and(iii) optionally a pharmaceutically acceptable excipient.

It is demonstrated herein that production of CHIKV-Δ5nsP3 by serialpassaging five or more times in Vero cells results in high levels ofsequence heterogeneity, particularly in the E2 structural protein (seee.g. Example 2 below). For instance, E168K and/or G55R mutations of theE2 protein often appeared by passage 5 (see e.g. Table 3 below), andboth correlated with a drop in immunogenicity. Accordingly, productionof CHIKV-Δ5nsP3 using five or more passages in Vero cells as describedin Hallengärd D, et al. 2014 supra can unfavorably result in high levelsof non-immunogenic mutants of CHIKV-Δ5nsP3 (such as E168K[E2]) in thevaccine composition. In contrast, it is demonstrated below that sequenceheterogeneity in the E2 structural protein after fewer than fivepassages was much lower (see e.g. Example 3—although the E168K mutationwas present after three passages, its frequency was only 18%).

Accordingly, in one aspect the pharmaceutical composition comprises (i)CHIKV-Δ5nsP3; and (ii) optionally a pharmaceutically acceptableexcipient; wherein at least 30% of the CHIKV-Δ5nsP3 particles present inthe composition express an E2 structural protein as defined by thepolypeptide sequence of SEQ ID NO: 2. In this embodiment, at least 30%of the CHIKV-Δ5nsP3 particles are non-mutants with respect to the E2structural protein, i.e. express the E2 structural protein of SEQ ID NO:2. In other words, the frequency of sequence heterogeneity (i.e. mutantCHIKV-Δ5nsP3 particles expressing at least one mutation in the E2structural protein of SEQ ID NO: 2) is 70% or less. Unless specifiedotherwise, when referring to “CHIKV-Δ5nsP3” or “CHIKV-Δ5nsP3 particles”in general it is intended to encompass both non-mutant and mutant formsof CHIKV-Δ5nsP3, i.e. CHIKV-Δ5nsP3 which express an E2 structuralprotein of SEQ ID NO: 2 and CHIKV-Δ5nsP3 which express an E2 structuralprotein having one or more mutations in SEQ ID NO: 2. In one embodiment,the E2 structural protein of the CHIKV-Δ5nsP3 contains no more thanabout ten point mutations. In one embodiment, the E2 structural proteinof the CHIKV-Δ5nsP3 contains no more than 9, 8, 7, 6, 5 or 4 pointmutations. In a preferred embodiment, the E2 structural protein of theCHIKV-Δ5nsP3 contains three or less point mutations, most preferablyonly one or two point mutations.

In preferred embodiments, at least 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the CHIKV-Δ5nsP3particles present in the composition are non-mutants, i.e. express an E2structural protein as defined by the polypeptide sequence of SEQ ID NO:2.

In one aspect, the pharmaceutical composition comprises (i)CHIKV-Δ5nsP3; and (ii) optionally a pharmaceutically acceptableexcipient; wherein less than 70% of the CHIKV-Δ5nsP3 particles presentin the composition express an E2 structural protein having one or moremutations with respect to the polypeptide sequence of SEQ ID NO: 2.

In one aspect, the pharmaceutical composition comprises (i)CHIKV-Δ5nsP3; and (ii) optionally a pharmaceutically acceptableexcipient; wherein less than 70% of the CHIKV-Δ5nsP3 particles presentin the composition express an E2 structural protein having the mutationE168K in the polypeptide sequence of SEQ ID NO: 2.

In a preferred embodiment, the mutations (e.g. the mutation E168K) inthe E2 structural protein are present at a frequency of 70% or less.e.g., less than 70% of the total CHIKV-Δ5nsP3 particles comprise one ormore mutations (or the mutation E168K) and 30% or more of the totalCHIKV-Δ5nsP3 particles express a non-mutated E2 structural protein or anE2 structural protein that does not comprise the mutation E168K.

In a preferred embodiment, less than 65%, 60%, 55%, 50%, 45%, 40%, 35%,30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the CHIKV-Δ5nsP3particles present in the composition express an E2 structural proteinhaving one or more mutations (such as, e.g., E168K) with respect to thepolypeptide sequence of SEQ ID NO: 2. For instance, the composition maycomprise 1-70%, 1-50%, 1-30%, 1-20%, 5-70%, 5-50%, 5-30%, 5-20%, 10-70%,10-50%, 10-30% or 10-20% of mutant particles (i.e. CHIKV-Δ5nsP3particles expressing an E2 structural protein having one or mutations(such as, e.g., E168K) with respect to the polypeptide sequence of SEQID NO: 2), compared to the total number of CHIKV-Δ5nsP3 particles(mutant and non-mutant) present in the composition. In one embodiment,the CHIKV-Δ5nsP3 particles expressing an E2 structural protein having anE168K mutation further comprise a mutation which mitigates the loss ofimmunogenicity conferred by the E168K mutation. In one embodiment, themutation is in the nsP1 protein, especially at residue A38. In apreferred embodiment, the CHIKV-Δ5nsP3 particles expressing an E2structural protein with an E168K mutation also express an nsP1 with anA38S mutation.

Also provided herein is a process for producing a pharmaceuticalcomposition of the invention, comprising the steps of 1) growing aCHIKV-Δ5nsP3 virus on a cell line, and 2) minimizing the presence ofimmunogenicity-reducing mutations of the CHIKV-Δ5nsP3 virus. In oneembodiment, the immunogenic CHIKV-Δ5nsP3 virus is propagated in a cellline selected from the group consisting of an EB66 cell line, a Verocell line, a Vero-αHis cell line, a HeLa cell line, a HeLa-S3 cell line,a 293 cell line, a PC12 cell line, a CHO cell line, a 3T3 cell line, aPerC6 cell line, a MDSK cell line, a chicken embryonic fibroblast cellline, a duck cell line and a diploid avian cell line. In someembodiments, said cell line is a duck cell line. In some embodiments,said cell line is a diploid avian cell line. In some embodiments, saidcell line is EB66 cell line. In a preferred embodiment, said cell lineis a Vero cell line.

In one embodiment, the presence of immunogenicity-reducing mutations isminimized by passaging CHIKV-Δ5nsP3 less than 5 times, preferably lessthan 4 times, preferably less than 3 times, preferably less than 2times, more preferably only one time, most preferably at most 3 times.As used herein, the passage numbers refer to the number of in vitropassages following virus rescue (P0). In a preferred embodiment, thevirus is passaged on Vero cells. In one aspect, the virus is grown at anoptimal temperature. In a preferred embodiment, said optimal temperatureis between about 28° C. and 37° C., preferably about 35° C.

In one embodiment, the host cell culture is infected with CHIKV-Δ5nsP3at an optimal MOI. In one aspect, an optimal MOI is defined as an MOIlow enough as to not require excessive amounts of working virus seedbank culture, but high enough to minimize immunogenicity-reducingmutations as described herein. In a preferred aspect, the optimized MOIis an MOI of less than 0.1, preferably an MOI of between about 0.1 and0.001, more preferably an MOI of between about 0.09 to 0.0011, even morepreferably an MOI of about 0.05 to 0.005, most preferably an MOI ofabout 0.01. In one aspect, the host cell confluency is assessed beforeinfection. In one aspect, the host cell confluency is between about 20and 90%, preferably between about 30 and 75%, more preferably betweenabout 40 and 60%, especially about 50 to 60%. In one aspect, the cellculture is infected at an optimal timepoint post-host cell seeding;i.e., at between day 2 and day 5 after host cell seeding, preferably atabout 4 days after host cell seeding. In one aspect, the virus particlesare harvested between day one and day 6 after host cell infection,preferably between day one and day 4, preferably on day one or day 2after host cell infection, preferably on both day one and day 2 afterhost cell infection.

In one aspect, immunogenicity-reducing mutations are point mutations atany location in the genome of CHIKV-Δ5nsP3 as defined by thepolynucleotide sequence of SEQ ID NO: 1. In one embodiment, theimmunogenicity-reducing mutations are present in the genome at alocation other than the E2 protein. In a preferred embodiment, theimmunogenicity-reducing mutations are located in the E2 protein,preferably at amino acid residues 55 and/or 168, e.g., G55R and/or E168Kmutations, especially E168K. In some embodiments, theimmunogenicity-reducing mutations as described herein are mitigated or“rescued” by other mutations in the genome. In one embodiment, amitigating mutation of E168K is an A38S mutation of nonstructuralprotein 1 (nsP1).

In one aspect, the frequency of the E168K mutation of the E2 protein ofthe CHIKV-Δ5nsP3 is less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, preferably less than 50% in the total pool of harvestedCHIKV-Δ5nsP3.

In one aspect, the current invention provides an immunogenicCHIKV-Δ5nsP3 obtainable by the process provided herein. In anotheraspect, the current invention provides a pharmaceutical compositioncomprising an immunogenic CHIKV-Δ5nsP3 obtainable by the processprovided herein.

Aspects of the invention provide a use of the process described hereinfor manufacturing a composition for immunization against a Chikungunyavirus infection. In a preferred embodiment, the composition is avaccine. In one embodiment, the vaccine is administered to the subjectonce, twice or three or more times. In one aspect, CHIKV-Δ5nsP3 viralparticles isolated from immunized subjects have a similar point mutationprofile to the vaccine composition administered, particularly withregard to point mutations in the E2 structural protein. In oneembodiment, the vaccine is administered once or twice. In a preferredembodiment, the vaccine is administered only once; e.g., a one-shotvaccine. In one aspect, a booster vaccination is optionallyadministered. In certain preferred aspects, the pharmaceuticalcomposition is provided in lyophilized form.

Other aspects provide compositions comprising the virus particlesobtainable by the process described herein for treating and/orpreventing a Chikungunya virus infection. In one aspect, thecompositions are for use in a method of stimulating an immune responsein a subject and/or in a method of treating or preventing a Chikungunyavirus infection. As used herein, the term “preventing” also means“protecting from”. The Chikungunya virus infection in one aspect may becaused by West African, East/Central/South African (ECSA) and/or Asiangenotypes of Chikungunya virus.

Virus preparations produced using any of the processes described hereinmay be further subjected to additional processing steps, includingadditional filtration steps and/or lyophilization. The virus preparationmay be subjected to analysis for purity of the preparation. For example,the virus preparations may be assessed for the presence of impuritiesand contaminants, such as, e.g., host cell genomic DNA, and/or host cellproteins. The purity of a virus preparation may be assessed using anymethod known in the art, such as size exclusion chromatography (SEC),optical density at different wavelengths, protein gel electrophoresis(e.g., SDS-PAGE), Western Blotting, ELISA, PCR, and/or qPCR.

In some embodiments, the virus preparation is assessed for residualimpurities or contaminants. In some embodiments, the amount of residualimpurities or contaminants is compared to the amount of impurities orcontaminants at an earlier stage in the purification process, such as,e.g., directly after viral harvest. In some embodiments, the relativereduction of impurities in the final virus preparation is between 60-95%relative to the presence of impurities at an earlier stage in thepurification process. In some embodiments, the relative reduction ofimpurities in the final virus preparation is approximately 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95%. In someembodiments, the final virus preparation contains less than 5%impurities or contaminants. In some embodiments, the final viruspreparation contains less than 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5,0.4, 0.3, 0.2, or less than 0.1% impurities. In a preferred embodiment,the final virus preparation contains less than 1% impurities.

Any of the processes described herein may be used in the manufacture ofa composition comprising purified virus for administration to a subject.In some embodiments, the subject is a mammalian subject, such as a humanor a non-human animal, including livestock, pets or companion animals.In some embodiments, the composition is administered to a subject inneed of immunization against the virus or similar virus as that of thevirus preparation. In some embodiments, the virus preparations orcompositions comprising viruses purified using the processes describedherein are for treating or preventing infection with the virus or asimilar virus as that of the virus preparation. In a preferredembodiment, the virus preparations or compositions comprising virusespurified using the processes described herein are for treating orpreventing a Chikungunya virus infection, particularly a Chikungunyavirus infection caused by West African, East/Central/South African(ECSA) and/or Asian genotypes of Chikungunya virus.

The CHIKV-Δ5nsP3 pharmaceutical compositions or CHIKV-Δ5nsP3 virusespurified using the processes described herein may be administered to asubject by any route known in the art. In some embodiments, thepreparations or compositions may be administered via conventionalroutes, such as parenterally or orally. As used herein, “parenteral”administration includes, without limitation, subcutaneous,intracutaneous, intradermal, intravenous, intramuscular, intraarticular,intraperitoneal, intrathecal or by infusion.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular and cellular biology, microbiology, virology, cell or tissueculture, genetics and protein and nucleic chemistry described herein arethose well-known and commonly used in the art. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well known in the art and as described invarious general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated.

TABLE A-1 Abbreviations Abbreviation Definition CHIKV Chikungunya virusCHIKV-Δ5nsP3 CHIKV with a defined deletion mutation in nsP3 (SEQ IDNO: 1) ECSA East-central south African E (1, 2, 3) proteins Envelopeproteins C protein Capsid protein 6K protein 6 kilodalton protein nsPNon-structural protein MEM Minimum essential medium EMEM Eagle's MEM FBSFetal bovine serum PBS Phosphate buffered saline Pfu Plaque forming unitPRNT Plaque reduction neutralization test NGS Next generation sequencingh hour d Day AA Amino acid PCR Polymerase chain reaction qPCRQuantitative PCR s.c. Subcutaneous TSE Transmissible SpongiformEncephalopathy p.i. Post-infection PP Polypropylene μF Micro-Farad μmmicrometer kDa kilodalton mg milligram RNA Ribonucleic acid Anti-antiAntibiotic-antimycotic SINV Sindbis virus SFV Semliki Forest virus WHOWorld Health Organization MVSB or P1-MVSB Master virus seed bank(Passage 1 after rescue) WVSB or P2-WVSB Working virus seed bank(Passage 2 after rescue) VRP Virus replicon particle TCID₅₀ Tissueculture infective dose LR-VRP VRP based on the La Reunion CHIKV isolateBHK(−21) Baby hamster kidney cells MOI Multiplicity of infection MMillion T75, T150, T175 T flask 75, 150, 175 cm² RB850 Roller bottle 850cm² Prob.F Probability as determined by the F-test in one-way analysisof variance ANOVA Analysis of variance DS Drug substance GLuc Gaussialuciferase GMP Good manufacturing practice R&D Research and developmentSEC Size exclusion chromatography SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis ELISA Enzyme-linked ImmunosorbantAssay w/w Weight/weight mL Milliliter CPE Cytopathic effect V Volts mMrnillimolar TBS Tris-buffered saline NaCl Sodium chloride LR2006-OPY1 LaReunion CHIKV isolate D-PBS Dulbecco's phosphate buffered saline RPMRotations per minute RRV Ross River virus MCB Master cell bank ATCCAmerican Type Culture Collection

EXAMPLES Example 1. Initial Trials for CHIKV-Δ5nsP3 Drug Substance (DS)Production

Assembly of Synthesized CHIKV-Δ5nsP3 Genome

The CHIKV-Δ5nsP3 virus genome was synthesized in five fragments at MWGEurofins (Germany) and was fully assembled in the pMA plasmid (pMXvector with ampicillin resistance), a standard cloning vector. The pMXvector backbone is shown in FIG. 1. All cloning and plasmid preparationprocedures were carried out under TSE-free conditions usingelectro-competent NEB10β E. coli cells. The cloning strategy used forthe full assembly of the CHIKV-Δ5nsP3 genome in pMA is outlined in FIG.3B. Briefly, the pMA plasmid containing fragment 1, which covers nsP1and part of nsP2 (as shown in FIG. 3A), was linearized via EcoRI/PacIrestriction digestion and fragment 2 covering parts of nsP2 and nsP3 wasfused to fragment 1. In parallel, fragment 3 (covering nsP4 and C) wasfused to fragment 4 (covering C and E2) via ClaI/PacI cloning. In athird cloning step, fragments 3 and 4 were cloned via AgeI/XhoI andfragment 5 via XhoI/PacI into the AgeI/PacI—linearized pMA alreadycontaining fragments 1 and 2. The cloning resulted in thepMA_CHIKV-Δ5nsP3 vector encoding CHIKV-Δ5nsP3, as verified bysequencing.

CHIKV-Δ5nsP3 Rescue from Vero Cells (“Virus Rescue”)

For the production of CHIKV-Δ5nsP3 virus particles from the engineeredpMA_CHIKV-Δ5nsP3 vector on Vero cells (virus rescue), thepMA_CHIKV-Δ5nsP3 plasmid was linearized by NotI restriction digestionand subjected to in vitro transcription using Ambion's mMessage mMachineSP6 Kit (AM130). RNA integrity was confirmed via gel electrophoresis(not shown). In parallel. Vero cells were prepared for electroporationwith viral RNA. Briefly, Vero cells were detached from cell cultureflasks using TrypLESelect (Gibco) and washed twice with PBS. Allcentrifugation steps were performed at 300 g at room temperature. ViralRNA was mixed with 8×10⁶ Vero cells in 800 μl PBS and the Vero cell/RNAmix was transferred into 0.4 cm electroporation cuvettes. Two pulseswere performed at 850 V, 25 μF, 200 Ohm. After electroporation. Verocells were kept at room temperature for 10 minutes and finallyresuspended in MEM/5% FCS/1% Antibiotic-Antimycotic (Anti-Anti)/2 mML-Glutamine and incubated in T75 flasks for 48 hours at 35° C./5% CO₂.Cell culture supernatant containing rescued CHIKV-Δ5nsP3 (passage 0; P0)was harvested and centrifuged at 3,000 g for 10 minutes at 4° C. Thevirus titer was determined by plaque and TCID₅₀ assay on Vero cells. Therescued CHIKV-Δ5nsP3 (P0) was stored at −80° C. The genomic structure ofthe thus obtained CHIKV-Δ5nsP3 virus vaccine candidate, also referred toas VLA1553, is shown in FIG. 2.

Verification of CHIKV-Δ5nsP3 Sequence

In order to verify the viral genome sequence, viral nucleic acids wereextracted from harvested cell culture supernatant using the QIAampMinElute Virus Spin Kit (QIAGEN #57704) and cDNA-synthesis was doneusing the SuperScript III First-Strand Synthesis System (LifeTechnologies, Catalog #18080-051) using random hexamers. PCR withPhusion High Fidelity Polymerase was performed with primers amplifyingoverlapping regions of the CHIKV-Δ5nsP3 genome, and the PCR productswere subjected to Sanger sequencing at MWG Eurofins, Germany. Thesequences of primer pairs used for PCR and sequencing are shown in Table1.

TABLE 1 Primer pairs used for CHIKV-Δ5nsP3 genome sequencing. PrimerForward primer sequence (5′-3′) SEQ ID Reverse primer sequence (5′-3′)SEQ ID Pair restriction sites (lower case) NO:restriction sites (lower case) NO: 1 ttaggatccGATGGCTGCGTGAGACAC 8taactcgagCCGTCAGGTCTGTTGAACAT 9 2 ttaggatccTACCACCAGGCGATTAAAG 10taactcgagCTTTGCCCACTTACTGAAGG 11 3 ttaggatccTGCTACAGAAGTCACGCC 12taactcgagGCCAAAGCGTGAATCAG 13 4 ttaggatccAACAGCTTGAGGACAGAGCG 14taactcgagCTCTGTCTCATCACGTCGG 15 5 ttaggatccAAATTGCAGTCATAGGAGTCTTC 16taactcgagAGTACGTTGACGTGCTCTGA 17 6 ttaggatccGTGGGTTAAACAACTGCAAA 18taactcgagGGTTAAAGTCTTCTATCCTCCTGG 19 7 ttaggatccGGATAACCACTGGGATAATAGG20 taactcgagAGTTGTGAAATTCCTTCTGCC 21 8 ttaggatccCCAGATAGAACCAGTGAAC 22taactcgagCAGCAGCTCTACTTGGGTC 23 9 ttaggatccAGGAGGGAAAGACAGGCT 24taactcgagCCCTCGCCTTCTTCTG 25 10 ttaggatccCAAAATAGAAGGAGTGCAAAAAG 26taactcgagCCTGGAGTTTCTTAAGTAATAGTTGC 27 11 ttaggatccACCGGTCCAGGTCATTTA 28taactcgagGCAGCAAATTCTTCCCAG 29 12 ttaggatccCCATTCCAGAACACACTACAG 30taactcgagATACCTGATTTCATCATGGC 31 13 ttaggatccCCTTTGATAAGAGCCAAGATG 32taactcgagTACAAAGTTATGACGGGTCCT 33 14 ttaggatccCAACGAACAGGGCTAATTG 34taactcgagGACCGCTTAAAGGCCAG 35 15 ttaggatccGTGCATGAAATCGAAAATG 36taactcgagTGGTCTTGTGGCTTTATAGACA 37 16 ttaggatccAACCGGAGGAAACCCTAC 38taactcgagGTACCGCACCGTCTGG 39 17 ttaggatccAGCTACCTTGCAGCACGT 40taactcgagCCCACATCGACAGG 41 18 ttaggatccCGAGCCGTATAAGTATTGGC 42taactcgagCGCCGGTGAAGACCTTAC 43 19 ttaggatccACTACTGTCAGTCACTTTGGAGC 44taactcgagTACCGGGTTTGTTGCTATTT 45 20 ttaggatccCACAACTGGTACTGCAGAGAC 46taactcgagGCGTAGCCCTTTGATCTATAG 47 21 ttaggatccGGTGCTATGCGTGTCGT 48taactcgagATCTCCTACGTCCCTGTGGG 49

Passaging of CHIKV-Δ5nsP3 on Vero Cells

Following rescue of CHIKV-Δ5nsP3, Vero cells were infected and the viruswas serially passaged in three replicates (see Table 2). For passaging.Vero cells seeded in T150 flasks and grown to confluency (1-3 days) werewashed twice with 1×DPBS before the addition of 20 mL infection medium(EMEM w/o serum). Inoculum was added directly to the flask at theindicated volume and the cells were incubated for 24 h at 35° C., 5%CO₂. Passaging was done in three replicates (A. B and C) with infectionof Vero cells at an MOI of 0.01 for the first passage from virus rescue(P0). The 20 mL harvests were transferred to a 50 mL PP tube 24 h p.i.,cell debris was removed by centrifugation (3000 g, 10 min) and thesupernatant transferred to a fresh 50 mL PP tube. A 49% (w/w) sucrosesolution was added to a final concentration of 10% (w/w) and 1 mLaliquots of stabilized harvest were stored at ≤−70° C.

Subsequent infections were carried out with harvest without sucrose. Theinfections were carried out using different volumes of harvest whichwere roughly calculated based on the observed cytopathic effect in theprevious passage and in parallel replicates. Volumes of harvest used forinfection varied between 5 μL and 1 mL. Infections were followed by asingle harvest after 24 h. Note that the MOI used for production ofpassages 2-16 was determined retrospectively after TCID₅₀ results wereavailable, resulting in a wide (“uncontrolled”) range of MOIs throughoutthe experiment (See Table 2). This procedure was performed up to 16passages in the three parallel replicates (replicates A. B and C)allowing systematic observation of various parameters during adaptationof CHIKV-Δ5nsP3 to Vero cell passaging. During this experiment, yield(TCID₅₀/mL), volume of infection, number of Vero cells per flask, Verocell passage number and cytopathic effect (CPE) were recorded. Themultiplicity of infection (MOI) was determined retrospectively based onthe measured TCID₅₀ and also recorded. The CHIKV-Δ5nsP3 passages weresimultaneously assessed for plaque size in all three replicates.

TABLE 2 Serial passaging of CHIKV-Δ5nsP3 on Vero cells following virusrescue, performed in triplicate. Data shown include harvest yield (24 h;TCID₅₀), infection volume (mL), number of cells/flask, Vero cell passagenumber, multiplicity of infection (MOI) and observed cytopathic effect(CPE) at 24 h post-infection. Replicate A Pas- TCID₅₀/ Infection Cells @Vero MOI CPE @ sage mL (mL) T150 passage (TCID₅₀) 24 h P0 9.77E+06 n.a.P1A 2.59E+07 0.02 2.00E+07 149 0.010 50 P2A 1.90E+07 1 2.90E+07 1490.893 50 P3A 2.92E+07 0.1 2.80E+07 149 0.068 20 P4A 2.14E+08 0.372.00E+07 150 0.540 90 P5A 1.01E+08 0.1 2.70E+07 150 0.792 60 P6A3.50E+08 0.1 2.60E+07 150 0.390 60 P7A 7.43E+08 0.037 1.30E+07 151 0.99750 P8A 8.02E+08 0.05 1.60E+07 151 2.323 90 P9A 1.51E+09 0.01 2.40E+07151 0.334 60 P10A 3.60E+09 0.005 1.98E+07 152 0.381 80 P11A 1.88E+090.005 1.84E+07 152 0.982 90 P12A 6.94E+08 0.005 1.30E+07 152 0.724 90P13A 4.22E+09 0.01 1.44E+07 153 0.482 60 P14A 3.05E+09 0.01 1.90E+07 1532.219 90 P15A 3.03E+09 0.01 2.20E+07 153 1.384 90 P16A 2.91E+09 0.011.70E+07 154 1.781 90 Replicate B Pas- Infection cells @ Vero MOI CPE @sage TCID₅₀/mL (mL) T150 passage (TCID₅₀) 24 h P0 9.77E+06 n.a. P1b6.47E+07 0.02 2.00E+07 149 0.010 50 P2B 5.32E+07 1 2.90E+07 149 2.232 50P3B 2.27E+07 0.1 2.80E+07 149 0.190 20 P4B 1.65E+08 0.08 2.00E+07 1500.091 80 P5B 1.65E+08 0.1 2.70E+07 150 0.613 60 P6B 4.06E+08 0.12.60E+07 150 0.636 60 P7B 9.19E+08 0.092 1.30E+07 151 2.875 60 P8B7.43E+08 0.1 1.60E+07 151 5.744 90 P9B 2.14E+09 0.025 2.40E+07 151 0.77475 P10B 4.06E+09 0.01 1.98E+07 152 1.080 90 P11B 2.09E+09 0.01 1.84E+07152 2.214 90 P12B 8.76E+08 0.01 1.30E+07 152 1.608 90 P13B 2.91E+09 0.011.44E+07 153 0.609 60 P14B 4.22E+09 0.01 1.90E+07 153 1.530 90 P15B3.79E+09 0.01 2.20E+07 153 1.917 95 P16B 3.79E+09 0.01 1.70E+07 1542.232 90 Replicate C Pas- Infection cells @ Vero MOI CPE @ sageTCID₅₀/mL (mL) T150 passage (TCID₅₀) 24 h P0 9.77E+06 n.a. P1C 3.24E+070.02 2.00E+07 149 0.010 50 P2C 3.29E+07 1 2.90E+07 149 1.117 50 P3C2.54E+07 0.1 2.80E+07 149 0.118 20 P4C 1.29E+08 0.15 2.00E+07 150 0.19190 P5C 4.13E+08 0.1 2.70E+07 150 0.477 60 P6C 5.91E+08 0.1 2.60E+07 1501.587 60 P7C 1.85E+09 0.185 1.30E+07 151 8.415 70 P8C 4.83E+08 0.21.60E+07 151 23.073  90 P9C 1.03E+09 0.1 2.40E+07 151 2.013 90 P10C5.14E+09 0.015 1.98E+07 152 0.783 90 P11C 1.51E+09 0.015 1.84E+07 1524.205 90 P12C 1.15E+09 0.015 1.30E+07 152 1.738 90 P13C 4.22E+09 0.011.44E+07 153 0.802 65 P14C 3.81E+09 0.01 1.90E+07 153 2.219 90 P15C1.07E+09 0.01 2.20E+07 153 1.730 90 P16C 2.48E+09 0.01 1.70E+07 1540.631 90

Trends Observed During Serial Passaging Under “Uncontrolled” MOIConditions

During CHIKV-Δ5nsP3 adaptation to Vero cell passaging, it was observedthat total CHIKV-Δ5nsP3 virus yield increased substantially withincreased passage number on Vero cells as shown in Table 2. As shown inFIG. 4A, passages 6 and above yielded an approximately 100-fold increasein titer compared with virus rescue (P0) and early passages. Alsoobserved was a concomitant decrease in CHIKV-Δ5nsP3 plaque size (FIG.4B). The effect of in vitro passaging of wild-type Chikungunya virus onplaque size in other cell lines has been previously described by GardnerC L, et al. (Gardner C L, et al., 2014, supra). A high virus yield wouldbe highly desirable for industrial production of the inactivated virus;however, it was also herein observed that CHIKV-Δ5nsP3 became lessimmunogenic with increased passaging as shown in FIG. 4C. Briefly, todetermine the immunogenicity of different passages of CHIKV-Δ5nsP3 asgenerated in Table 2, above, groups of five C57Bl/6 mice were immunizedonce subcutaneously with a dose of 10⁵ TCID₅₀ CHIKV-Δ5nsP3 passage 5(P5B), passage 8 (P8B) or P15 (P15C). CHIKV-Δ5nsP3 at P0 (virus rescue),also at 10⁵ TCID₅₀, was used as a positive control. Day 21 serum poolswere assessed for their capacity to neutralize CHIKV-Δ5nsP3 (P0) in aPRNT assay on Vero cells, by testing 4-fold serial serum dilutionsranging from 1:20 to 1:327.680. As shown in FIG. 4C, the immunogenicityin mice of the P5B CHIKV-Δ5nsP3 showed slightly shifted immunogenicitycompared with the unpassaged CHIKV-Δ5nsP3 (P0), whereas a P8B virusshowed substantially reduced immunogenicity comparable to the P15Cvirus. (As the P15C virus was non-immunogenic, it served as a negativecontrol in subsequent PRNT assays.)

Finally, selected passages of the CHIKV-Δ5nsP3 were tested for geneticstability by Sanger sequencing. Upon passaging of CHIKV-Δ5nsP3 on Verocells up to 16 times, it was verified that the 60 amino acid deletion inthe nsP3 gene responsible for the attenuation of the virus wasgenetically stable, indicating that the virus does not revert back towild-type, an important safety consideration for live attenuatedvaccines.

Trends Observed During Serial Passaging Under Controlled MOI Conditions

The use of high MOIs (e.g., higher than 0.1) is not conducive to anindustrial scale process as too much starting material is needed. Inthis regard, the use of a lower MOI (0.01) over three passages wastested and the immunogenicity of the resulting passages was determined.As shown in FIG. 5A, three passages from Replicate B in Table 2, werepassaged under uncontrolled conditions, with MOIs ranging from 0.01 to2.23. From the thus-obtained passage 3 (P3B), further passages were doneup to P6 using an MOI of 0.01 for each passage and shown as “controlledconditions at MOI 0.01” in FIG. 5A. Under conditions using an MOI of0.01, the immunogenicity of the resulting virus was lost very quickly asshown in FIG. 5B. By passage 5, the CHIKV-Δ5nsP3 was renderednon-immunogenic. This result was in contrast to the results seen withP5B in FIG. 4C, which was produced with a much higher MOI (see Table 2.Replicate B) and was still immunogenic. This observation seemed toindicate that a lower MOI leads to faster selection during Vero cellpassaging for mutations that affect the immunogenicity of CHIKV-Δ5nsP3.

Example 2. Defining Sequence Heterogeneities of CHIKV-Δ5nsP3 whichAffect Immunogenicity

Due to the observed reduction/loss of immunogenicity (neutralizingantibody titer) and decreased plaque size at higher CHIKV-Δ5nsP3passages, it was of interest to analyze possible sequenceheterogeneities within the viral populations at different passagenumbers. In addition, it was of interest to analyze the sequence ofindividual plaques of the viral population. Unpassaged CHIKV-Δ5nsP3 (P0)did not show sequence heterogeneities based on Sanger sequencing. Ingeneral, with increased passage numbers an increase in sequenceheterogeneities for all 3 replicates was observed (Replicates A, B andC; Table 3). In the case of passaging replicate C at passage 8 (P8C),the virus population was still heterogeneous (sequence heterogeneitiesshown in Table 3), whereas the P15C passage showed a more homogenousvirus population with defined point mutations (indicated by *). Theimmunogenicity data shown in FIG. 4C (Example 1, above) focused on P5Band P8B, which showed sequence heterogeneities in the CHIKV-Δ5nsP3non-structural proteins (nsP) and envelope protein E2 as shown in Table3 below.

TABLE 3 Sequence heterogeneities in CHIKV-Δ5nsP3 at passages P5, P8 andP15 (Replicates A, B and C as produced in Table 2). Sequenceheterogeneities were determined by Sanger sequencing. For that purpose,viral nucleic acids were extracted from harvested cell culturesupernatant using the QIAamp MinElute Virus Spin Kit (Qiagen). The cDNAwas synthesized using the SuperScript III First-Strand Synthesis System(ThermoFischer) using random hexamers. PCR with Phusion High FidelityPolymerase was performed with primers amplifying overlapping regions ofthe CHIKV-Δ5nsP3 genome (Table 1), which were sequenced by Sangersequencing at MWG Eurofins, Germany. The readout shows results ofautomated base calling (>20%) as well as heterogeneities detected byvisual analyses of sequencing chromatograms. *Full mutations (i.e.,100%) are indicated by an asterisk. Replicate A Replicate B Replicate CP5A P8A P15A P5B P8B P15B P5C P8C P15C Gene Sequence HeterogeneitiesnsP2 G577W G577W G577W L494I G577W G577W* G577W G621R nsP3 R470S R470SnsP4 V113I E2 E168K G55R G55R E168K G55R G55R G55R G55R G55R* E247KT230I M171V A423A H99Y H99Y E247K T230I T230I* A423A H232Y T230I E168KE168K E247D E247D H232Y T230I T230I E247D

Expansion and Sequencing of Single CHIKV-Δ5nsP3 Plaques

To understand the effect of individual mutations on immunogenicity andconsequently develop a controlled and reproducible production processfor a highly immunogenic CHIKV-Δ5nsP3 vaccine, individual plaques fromCHIKV-Δ5nsP3 isolates P5B and P8B were picked. Briefly, serial dilutionsof P5B and P8B CHIKV-Δ5nsP3 were used for infection of Vero cells in aplaque assay (described under FIG. 4) and after an incubation of 72hours, single plaques of different morphologies were picked (smallplaques were preferentially picked, since they began to appear afterseveral passages on Vero cells) and expanded via re-infection of Verocells (5×10⁵) in 6-well plates. The different CHIKV-Δ5nsP3 samples thatderived from single plaques were selected based on mutations in the E2gene sequence prior to one further expansion on Vero cells. ClonesP5B-02, P5B-03, P5B-04, P5B-07, P5B-11, P8B-01 and P8B-05 were expandedand purified for in vivo immunogenicity experiments. Individual plaqueswere expanded on Vero cells grown in Roller Bottles using a single 850cm² Roller Bottle with CellBIND surface for each isolate. Upon reachingconfluency, cells were washed twice with 100 mL D-PBS+Ca+Mg before 100mL infection medium (EMEM w/o serum) containing the inoculum at an MOIof 0.01 was added and the cells were incubated for 24 h at 35° C., 5%CO₂. The 100 mL harvests were transferred to 50 mL PP tubes 24 h p.i,and the cell debris was removed by centrifugation, followed by a 0.2 μmfiltration using Steriflip® vacuum filtration devices (Merck).Individual clarified virus harvests were first concentrated usingAmicon® Centrifugal Filter devices, diafiltrated to TBS buffer andpurified by protamine sulphate and Capto™ Core700 treatment. PurifiedCHIKV-Δ5nsP3 clones were then used for further studies.

The full genome sequences of the expanded CHIKV-Δ5nsP3 samples, P5B+1and P8B+1; namely P5B-02, P5B-03, P5B-04, P5B-07, P5B-11, P8B-01 andP8B-05 as described above, derived from single plaques, P5B and P8B,respectively, were assessed by Sanger sequencing. The observed pointmutations of the individual plaques are summarized in Table 4 andschematic genomic sequences are shown in FIGS. 6B and 6D.

In order to assess the effect of specific point mutations on theimmunogenicity of CHIKV-Δ5nsP3, day 19 mouse sera from mice immunizedwith the individual plaque-derived viruses were generated and analyzedin PRNT. Briefly, a single dose of CHIKV-Δ5nsP3 at an intended TCID₅₀dose of 10⁵ was administered subcutaneously to C57Bl/6 mice (10 pertreatment group) and pools of day 19 sera were analyzed in a PRNT assayat 4-fold serial dilutions ranging from 1:20 to 1:327,680 for theirvirus neutralization capacity. The virus that was neutralized in thePRNT corresponded to a passage 2 CHIKV-Δ5nsP3 (P2, 560 pfu/ml) which didnot show sequence heterogeneities and therefore was identical insequence to the unpassaged CHIKV-Δ5nsP3 (P0). The neutralization mix(560 pfu/ml CHIKV-Δ5nsP3 P2 and serial serum dilutions) was incubatedfor 1 hour at room temperature and added onto Vero cells, followed byincubation for 2 hours. Finally, a methylcellulose overlay (0.8%) wasadded followed by incubation for 72 hours. The plaque readout was donefollowing crystal violet staining (0.5% crystal violet in 5%Formaldehyde).

TABLE 4 Mutations in single picked plaques of P5 and P8 CHIKV-Δ5nsP3passages. Sequencing of the full CHIKV-Δ5nsP3 genome was performed atMWG Eurofins, Germany, using the primers in Table 1. All P5B and P8Bclones corresponded to a P6 (P5B + 1) and P9 (P8 + 1) CHIKV-Δ5nsP3,respectively, expanded on Vero cells. The PRNT₅₀ titers were calculatedin GraphPad Prism using non-linear − 3 parameter calculations. The viralprotein in which the respective point mutations were identified areshown in brackets. PRNT₅₀ values for non-immunogenic isolates were notmeasurable as indicated. Experiment # Isolate Passage Point mutation(s)Immunogenicity PRNT₅₀ 4399gr1 P0 #1  0 N/A Positive control  386 4415gr1P0 #2  0 N/A Positive control 4215 4415gr2 P5B-02 5 + 1 E168K[E2] + A38S[nsP1] retained  373 4415gr3 P5B-03 5 + 1 H232Y[E2] retained  1124415gr4 P5B-04 5 + 1 E168K[E2] lost — 4415gr5 P5B-07 5 + 1 E168K[E2]lost — 4415gr6 P5B-11 5 + 1 E247K[E2] retained  219 4415gr7 P8B-01 8 + 1H99Y/E168K[E2] + R470S[nsP3] lost — 4415gr8 P8B-05 8 + 1G55R/H232Y[E2] + G577W[nsP2] lost — 4399gr2 P15C-DS 15 G55R/T230I[E2] +G577W[nsP2] Negative control — — = not measurable

As can be seen in FIG. 6A, the immunogenicity of CHIKV-Δ5nsP3 P5B-11 andP5B-03, both with single point mutations in the E2 protein, E247K andH232Y (FIG. 6B), respectively, is not affected when compared to theimmunogenicity of P0 CHIKV-Δ5nsP3. The neutralization capacity of P0serum is shown from two independent mouse experiments (4415; P0 #2 and4399; P0 #1) delineating the acceptable range of immunogenicity. On theother hand, P8B-05, characterized by 3 point mutations, G577W, G55R andH232Y in nsP2 and E2, respectively, is non-immunogenic in mice. The G55Rmutation was already described in literature by Gardner C L, et al., asbeing a result of passaging CHIKV in vitro. The viral particle isaffected by an increased dependence on heparan sulfate binding in vitrowhich leads to an attenuation in vivo (Gardner C L, et al., 2014,supra).

As can be seen in FIG. 6C, only one of the four CHIKV-Δ5nsP3 samplesderived from single plaques (P5B-02) was still immunogenic and wascharacterized by two point mutations. A38S and E168K in nsP1 and E2(FIG. 6D), respectively. Also, P8B-01 with three point mutations, R470Sin the nsP3 and H99Y and E168K in the E2 protein, was non-immunogenic inmice and was comparable to the negative control P15C. P5B-04 and P5B-07each had single point mutations in E2, i.e., Glutamic acid 168 wasmutated to a Lysine (E168K), which was also shown to have a directeffect on immunogenicity, since CHIKV-Δ5nsP3 neutralization capacity waslost.

In summary, it was observed that many of the mutations arising duringpassaging on Vero cells were located in the E2 protein. Some of theidentified point mutations in the E2 protein and/or other parts of thegenome did not substantially affect immunogenicity of the virus;particularly the H232Y[E2] and E247K[E2] mutations. However, some of theother identified mutations in the CHIKV-Δ5nsP3 resulted in loss ofimmunogenicity; particularly the frequently-occurring E168K[E2]mutation, whether alone or in combination with other mutations. Aninteresting exception was the mutant with both E168K[E2] and A38S[nsP1]mutations, which maintained immunogenicity. This observation suggeststhat the A38S[nsP1] mutation has a mitigating effect on the reducedimmunogenicity conferred by the E168K[E2] mutation. Furthermore, anisolate with G55R/H232Y[E2] and G577W[nsP2] mutations also demonstratedpoor immunogenicity, perhaps mainly due to the G55R mutation in E2, asthe H232Y mutation alone had little effect (see P5B-03).

The E168K and G55R mutations in Chikungunya virus E2 protein werepreviously described as conferring increased positive surface charge,leading to increased interaction with heparan sulfate and/or otherGlycosaminoglycans (GAGs), ultimately resulting in increased specificinfectivity. On the background of wild-type CHIKV, the mutations wereshown to cause a smaller plaque size, due to lower spread on platesmediated by binding to heparan sulfate. Furthermore, the mutationsresulted in attenuation of CHIKV in a mouse model of musculoskeletaldisease (MSD), with decreased spread in mice to organs and thus lowerlevels of viremia (Gardner C L, et al., 2014, supra; Silva L A, et al.,A single-amino-acid polymorphism in Chikungunya virus E2 glycoproteininfluences glycosaminoglycan utilization (2014) J Virol.;88(5):2385-97). The fact that the presence of E168K and G55R mutationsin an otherwise wild-type CHIKV resulted in intermediate attenuation isconsistent with the present disclosure with regard to reduced plaquesize or reduced immunogenicity in vivo. However, it was unexpected thatthe said two mutations on the background of the attenuated CHIKV-Δ5nsP3would result in loss of immunogenicity in mice as reported herein.

It has also been reported as a common phenomenon for other cell culturepassaged alphaviruses such as Sindbis virus (SINV; Klimstra W B, et al.,Infection of neonatal mice with Sindbis virus results in a systemicinflammatory response syndrome (1999) J. Virol.; 73(12):10387-98;Klimstra W B, et al., The furin protease cleavage recognition sequenceof Sindbis virus PE2 can mediate virion attachment to cell surfaceheparan sulfate (1999) J. Virol.; 73(8):6299-306; Byrnes and Griffin.Binding of Sindbis virus to cell surface heparan sulfate (1998) J.Virol.; 72(9):7349-56), Ross River virus (RRV; Heil M L, et al., Anamino acid substitution in the coding region of the E2 glycoproteinadapts Ross River virus to utilize heparan sulfate as an attachmentmoiety (2001) J. Virol.; 75(14):6303-9) and Semliki Forest virus (SFV;Smit J M, el al., Adaptation of alphaviruses to heparan sulfate:interaction of Sindbis and Semliki forest viruses with liposomescontaining lipid-conjugated heparin (2002) J. Virol.; 76(20):10128-37)that substitutions for positively-charged residues in E2 confer enhancedheparan-sulfate dependent infectivity in vitro and that these mutationscan be selected within a few serial in vitro passages. Further, it wasshown that such mutations led to attenuation of the viruses in vivo(Byrnes A P and D E Griffin, Large-plaque mutants of Sindbis virus showreduced binding to heparan sulfate, heightened viremia, and slowerclearance from the circulation (2000) J. Virol.; 74(2):644-51; KlimstraW B, et al. 1999, supra).

Because sequence heterogeneities, with a concomitant drop inimmunogenicity, were already apparent at passages P5 and P8 ofCHIKV-Δ5nsP3, sequence heterogeneities at earlier passages, as well astheir effects on immunogenicity, were examined more closely as outlinedbelow.

Example 3. Defining Sequence Heterogeneities and Immunogenicity ofCHIKV-Δ5nsP3 at Passage P3

The occurrence at later passages of sequence heterogeneities withadverse effects on the immunogenicity of CHIKV-Δ5nsP3 as measured byneutralizing antibody titers warranted finding the optimal passage whichwas characterized by both high immunogenicity as well as a viral titersufficient for production of an effective vaccine.

To determine genetic stability of the CHIKV-Δ5nsP3 during MVSB (P1),WVSB (P2) and CHIKV-Δ5nsP3 drug substance (“VLA1553”) (P3) production,independently generated passages 1, 2 and 3 were sequenced. Asdetermined by Sanger sequencing, P0 (virus rescue), P1 (MVSB) and P2(WVSB) did not show any obvious sequence heterogeneities. The next stepwas to demonstrate reproducibility of genetic stability of P3 derivedpurified drug substance (DS) using P2 (WVSB) for infection. In total,four independent P3 harvests, consisting of combined day 1 and day 2harvests, were produced in two T150 T-flasks using P2 (WVSB) forinfection (MOI 0.01). For each replicate, the individual harvests at day1 and day 2 were pooled (total volume ˜50 mL) and concentratedapproximately 10-fold (Amicon 100 kDa ultrafiltration device).Diafiltration was done against 25 mM Tris/150 mM NaCl. pH 7.4, followedby protamine sulfate treatment (2 mg/mL final concentration) toprecipitate host cell DNA. The clear supernatant was then furtherpurified by batch adsorption chromatography using CaptoCore 700 resin(addition of ˜1 mL of 50% slurry in Tris/NaCl buffer). The resin wasremoved by centrifugation and sucrose was added to a final concentrationof 10% to allow freezing and thawing of CHIKV-Δ5nsP3. The finalformulation was then 0.2 μm sterile filtered and stored frozen (<−65°C.) until further processing.

At passage 3 (P3), no heterogeneities by automatic base calling weredetected (Eurofins—all <20%). However, by visual inspection, a smallfraction of the viral population showed a consistent increase in theE168K and E247K sequence heterogeneities in the gene for the CHIKVglycoprotein E2, which was absent in the rescued CHIKV-Δ5nsP3 (P0) aswell as the MVSB and WVSB samples. FIG. 7 shows sequencing chromatogramsfor the four independently generated P3 DS samples (Examples 1-4)compared with the virus at passage 0 (P0). As indicated by the boxoutlines, a G/A heterogeneity at the codon for amino acid 168 (genomicnucleic acid position 8882) was detectable in all four replicates andverified by the reverse sequencing reaction, which revealed the sameheterogeneity. The same position in the P0 sample showed a sharp peakfor G in the forward sequencing reaction (and C in the reversesequencing reaction). The E247K heterogeneity (genomic nucleic acidposition 9119), on the other hand, was present but barely detectable inthe sequencing chromatograms.

Additionally, next generation sequencing of P3 was carried out andcompared with sequencing of passage 1 (P1-MVSB) in order to quantify theamount of E168K and E247K within the viral population. As can be seen inFIG. 8, at the stage of P1-MVSB, only a very low level of sequenceheterogeneities was detectable (all below background; 15% cut-offindicated by the dotted line). The two representative P3 sample,however, showed an overall increase in sequence heterogeneities, withtwo mutations in the E2 gene that reached or rose above background(E168K in 18% and 15% of the viral population—genomic nucleic acidposition 8882 and, to a lower extent, E247K—genomic nucleic acidposition 9119).

In summary, the presence of an E168K mutation in the E2 protein ofCHIKV-Δ5nsP3 was identified by Sanger sequencing and NGS in eightindependently-generated P3 samples, demonstrating the reproducibility ofthis result. Representative sequencing examples are shown in FIGS. 7 and8. These data demonstrate that the emergence of an E168K mutation in theE2 protein of CHIKV-Δ5nsP3 virus was highly reproducible upon passagingin Vero cells and that it diminished the immunogenicity of theattenuated CHIKV-Δ5nsP3 virus as shown in Example 2 where P5Bplaque-derived viruses (P5B-04 and P5B-07 in FIG. 6) werenon-immunogenic. Two additional mutations, G55R and G82R, which wereobserved during passaging of CHIKV-Δ5nsP3 on Vero cells, like E168K havebeen reported to affect the virulence of CHIKV (Gardner C L, et al.,2014, supra. Silva L A, et al., 2014, supra and Gorchakov R, et al.,Attenuation of Chikungunya virus vaccine strain 181/clone 25 isdetermined by two amino acid substitutions in the E2 envelopeglycoprotein (2012) J. Virol.; 86(11):6084-96; Epub 2012/03/30), leadingto attenuation of the virus. Since these two mutations also negativelyimpacted the immunogenicity of CHIKV-Δ5nsP3, their emergence should beavoided for the production of a highly immunogenic CHIKV-Δ5nsP3 vaccinecandidate.

The locations of amino acids prone to mutation within the E1/E2 dimerare shown in FIG. 9. The three E2 amino acids G55, G82 and E168 are allaccessible on the surface and therefore their mutation may potentiallyaffect interactions of CHIKV with its cellular receptors. In contrast.E247[E2] is more buried within the protein structure and thus may not besurface exposed to the same extent. The position of E247 may be onereason why the E247K mutation did not noticeably affect theimmunogenicity of CHIKV-Δ5nsP3 as shown above.

Example 4. Determining the Threshold of the E168K Mutation for Loss ofImmunogenicity of a Heterogeneous CHIKV-Δ5nsP3 Virus Population

The above observations indicated that the E168K[E2] mutation appearsearly and frequently during passaging of CHIKV-Δ5nsP3 on Vero cells andis associated with lost immunogenicity. In order to develop a processfor the reliable manufacture of an effective, immunogeniclive-attenuated Chikungunya virus vaccine, the tolerance for thismutation in a sample of the CHIKV-Δ5nsP3 vaccine was tested by preparingdifferent ratios of the P3 drug substance and the virus P5B-07 (E168Ksingle mutant; see Table 3).

Passage 3 (P3) drug substance, which displayed about 20% E168Kheterogeneity (data not shown), was mixed with a preparation ofCHIKV-Δ5nsP3 from the P5B-07 isolate (E168K mutant) at ratios of 1:0.1,1:1 and 1:10. The mixtures were sequenced to verify the approximatefrequency of the E168K mutation in each virus preparation. As shown inFIG. 10 (see row labeled heterogeneity), the relative amount ofnucleotide A, compared with the wild-type nucleotide G, was shown toincrease with increasing levels of added P5B-07 isolate (up to 100% A inthe E168K control group 7).

In order to determine the effect of the E168K mutation onimmunogenicity, C57Bl/6 mice were immunized s.c. with the differentCHIKV-Δ5nsP3 samples specified in FIG. 10 with an intended dose of 3×10⁴TCID₅₀ (actual dose shown in FIG. 11D). Day 21 mouse sera were collectedand individual sera from the 1:0.1, 1:1 and 1:10 groups were tested andcompared with pooled sera from the two control groups, P3 (Group 6) andP5B-07 (E168K; Group 7) using a virus replicon particle (VRP)—basedneutralization assay (FIG. 11). VRPs resemble a replication deficientwild-type CHIKV displaying capsid and envelope proteins of theLR2006-OPY1 virus. The method was carried out essentially as describedby Gläsker S. et al. (Virus replicon particle based Chikungunya virusneutralization assay using Gaussia luciferase as readout (2013) Virol.J.; 10:235). The VRPs were produced by co-transfecting BHK-21 cells witha CHIKV replicon expressing Gaussia luciferase (GLuc) and two helperRNAs expressing wild-type CHIKV capsid protein and the remainingstructural proteins (E3, E2, 6K and E1), respectively. The resultingsingle round infectious particles were used in the CHIKV neutralizationassay using secreted GLuc as a readout. Upon neutralization of VRPs inthe Luciferase assay, a reduction of GLuc expression by BHK-21 cells canbe measured. It was observed that the capacity for elicitingneutralizing immunity dropped with higher amounts of the E168K mutant(FIGS. 11A, B and C, respectively). The number of individual miceshowing either positive, low positive or negative immune responses toChikungunya virus is shown in FIG. 11D.

This finding confirms that, as the ratio of E168K mutant to wild-typeviral particles in a virus population increases, the immunogenicity ofCHIKV-Δ5nsP3 in mice is diminished. It is therefore crucial to closelymonitor position E168 in E2 to ensure high immunogenicity of theCHIKV-Δ5nsP3 vaccine. Based on previous passaging processes andquantification of E168K within the viral population at passage 8, it wasobserved that at a rate of about 70% of the E168K mutation within theCHIKV-Δ5nsP3 population, the immunogenicity was lost when analyzingmouse serum pools in PRNT.

Example 5. Upstream Process for Reducing E168K Mutations in CHIKV-Δ5nsP3

The aim of this example was to characterize an optimized Vero cellculture based process for the production of CHIKV-Δ5nsP3 in rollerbottles. The impact of several upstream process parameters (MOI, day ofVero cell infection following plating and incubation temperature) onviral productivity and sequence heterogeneity of the E2 protein weretested using the GMP Working Virus Seed Bank (GMP WVSB B3005044: passage2, also referred to herein as “P2 CHIKV-Δ5nsP3”) and the R&D Veroworking cell bank to produce drug substance (DS; passage 3, i.e. alsoreferred to herein as “P3 CHIKV-Δ5nsP3”).

Preparation of GMP WVSB B3005044

A characterized Pre-Master Virus Seed Bank (PMVSB, Pre-Master Virus SeedBank AFR886/197579 from virus rescue from Vero cells) was establishedunder R&D conditions and a Pre-Master Virus Seed Bank was used togenerate the Master Seed Banks of the CHIKV-Δ5nsP3 under GMP conditions.The GMP Working Virus Seed Bank, VLA78-1553-WVSB-2016, batch B3005044was produced at Halix B.V. under the same production method and GMPconditions as described for the VLA78-1553-MVSB-2016, batch #B3005567.Briefly, the VERO Working Cell Bank (internal designation: ICB 2014/002)was expanded in four stages in a seed train using T75 cm² flasks (1×),then T175 cm² flasks (3×) and in the last stage 6×850 cm² roller bottlesas shown in FIG. 12. Four of the six roller bottles were used forproduction of the CHIKV-Δ5nsP3 seed banks. The total amount of cells inthe four roller bottles was determined. The Pre-Master Virus Seed Bankwas used to generate the CHIKV-Δ5nsP3 Master Virus Seed Bank.Subsequently, the CHIKV-Δ5nsP3 Master Virus Seed Bank was used togenerate the CHIKV-Δ5nsP3 Working Virus Seed Bank. Infection was done atall stages at an MOI of 0.01. After 24 hours of infection (35° C.; 5%CO₂; 0.5 RPM) the CHIKV-Δ5nsP3 was harvested. Tris and sucrose stocksolution was added to a final concentration of 5% Sucrose and 25 mM Trisin the harvest pool. After formulation, the pool was filtered through asteam sterilized 0.22 μm filter into a sterile 500 mL bio processcontainer by using a peristaltic pump. Filling of vials was done one byone with 0.7 mL formulated and filtered harvest using a peristalticfilling pump. A ThermoFisher capper/decapper device was used to open andclose the 2 D Matrix Cryo vials. Filled vials of the GMP seed banks werestored at <−65° C.

Culture of Vero Cells

Culturing of Vero cells was performed at 35° C. and 5% CO₂ in T75 cm²(T75), T175 cm² (T175) T-Flasks and 850 cm² roller bottles (850RB). Verocells used in the different experiments were derived from the GMP mastercell bank MCB ICB/2014/001. The internal designation of this researchworking cell bank was Bk5685. The GMP master cell bank was derived fromthe WHO Vero cell bank 10-87 P134 which originated from the InstitutMerieux (Aventis Pasteur) P129 bank and ultimately from the originalATCC CCL 81 P113 bank. More detail regarding the cell culture train isshown in FIG. 12. Cells were maintained in MEM medium supplemented with10% FBS and 2 mM L-glutamine.

Virus Production in 850 cm² Roller Bottles

Following two, four or five days of cell expansion at 35° C. in 850RB,cells were washed with PBS and infected with the CHIKV-Δ5nsP3 (WVSBB3005044) at MOIs of 0.1, 0.01 or 0.001 TCID₅₀/cell. For virusproduction, infected cells were incubated at 37° C., 35° C. or 28° C. in100 mL of MEM medium supplemented with 2 mM glutamine.

Virus Titration

Virus titers were determined on Vero cells using the TCID₅₀ assay. Cellswere seeded in microplates and infected with 10-fold serially dilutedvirus samples in EMEM supplemented with 0.5% FBS and 2 mM glutamine.After a one week incubation at 35° C./5% CO₂, virus-induced cytopathiceffects were monitored and viral titers were calculated according to theReed and Muench method (Reed, L. J.; Muench, H. A simple method ofestimating fifty percent endpoints (1938) The American Journal ofHygiene 27:493-497).

Virus Genome Extraction and Sequencing

Viral nucleic acid was extracted and purified from Vero cell culturesupernatant at the indicated timepoints using QIAamp MinElute Virus SpinKit (Qiagen) and cDNA synthesis was performed using SuperScript IIIFirst-Strand Synthesis System (ThermoFischer) using random hexamers. Forsequencing of the E2 gene region, first, PCRs with Phusion High FidelityPolymerase (ThermoFischer) were done using primers 16F, 16R, 17F, 17R,18F and 18R (for primer sequences see Table 1) to amplify overlappingregions of the CHIKV E2 gene. After purification of PCR amplicons,Sanger sequencing was performed at MWG Eurofins, Germany. In addition toanalyses of sequence heterogeneities that were detected by automaticbase calling (>20%), all sequencing chromatograms were manually read todetect also heterogeneities below the detection limit (<20%).

Optimization of a Process for Producing an Immunogenic P3 CHIKV-Δ5nsP3Drug Substance

To optimize the process for producing passage 3 CHIKV-Δ5nsP3 on Verocells, different MOIs, times of Vero cell infection post-seeding andtemperatures of incubation were tested in all combinations as shown inTable 5. Additionally, yields were analyzed at different days followinginfection. Three aspects of the harvested virus were monitored: viralproductivity, stability of the titer as well as the level of sequenceheterogeneity of the E2 structural protein.

TABLE 5 Parameters tested for production of CHIKV-Δ5nsP3 on Vero cellsin 850 cm² roller bottles and the parameters of the identified optimizedprocess. Optimized Tested* Process MOI (TCID₅₀/cell) 0,1, 0.01, 0.0010.01 Time of cell infection (post cell seeding) D2, D4, D5 D4Temperature (° C.) 37, 35, 28 35 *all combinations were performed.

Virus Production

CHIKV-Δ5nsP3 production kinetics achieved for all the conditions testedare shown in FIG. 13. The optimal process currently used to produceCHIKV-Δ5nsP3 (35° C. MOI 0.01, infection on day 4 after Vero cellseeding) was also included in the experiment, showing virus productivityat the expected level of 10^(8.0-8.5)/mL 24 h after infection (FIG.13B).

Compared to MOI and time of infection, temperature had the most impacton viral production kinetics. At 37° C. and 35° C. (FIGS. 13A and 13B,respectively), maximum productivity was achieved at day onepost-infection. The CHIKV-Δ5nsP3 infectivity dropped substantiallyduring the subsequent 24 hours, with a slightly less pronounced viraltiter loss at 35° C. Temperature reduction to 28° C. resulted in astriking change of the viral kinetics. Maximal viral productivity wasdelayed one to four days depending on the MOI used; and significantlyimproved virus titer stability was observed (FIG. 13C). The total virusyield produced from all of the harvests is shown in FIG. 14. For the 28°C. condition as shown in FIG. 13A, the yield of two harvests werecombined (day 1 and day 2 post-infection); for the 35° C. condition(FIG. 13B), the yield of all three harvests were combined (days 1-3) andfor the 37° C. condition (FIG. 13C), the yield of five harvests (days1-5) were combined.

To complete initial observations, viral productivity and titer stabilitydata were analyzed using a response surface quadratic model (FIGS. 15and 16). For viral productivity, the maximum virus titer values wereused. For virus titer stability, the delta value resulting from thesubtraction of the maximum titer from the titer measured one day laterin the production process was calculated and used. For total virusproductivity, the individual titers at each time point were added.

With ANOVA analysis of both models, it was possible to indicate thestatistically significant influencing factors (FIGS. 15A and 16A).Confirming previous observations, temperature was the strongest factoraffecting viral productivity and stability. In particular, lowtemperatures (28° C.) enabled higher viral titers while keeping thevirus titer reasonably stable over time. However, there was not asignificantly higher overall total virus productivity at thistemperature compared with 35° C. (see esp. FIG. 14).

Time of infection after Vero cell seeding also influenced the response,but to a lower extent. MOI did not have a significant impact. For bothmodels, infection at 72 h post cell seeding was an adequate time forcell infection. Conversely, a single temperature did not allow combiningoptimal virus production and titer stability since the highest viralyields were found at 35° C. and the most stabilized titers were observedat 28° C. (FIGS. 15B and 16B). Maximumal total virus productivity withinthe shortest time post infection was achieved at 35° C. (see FIG. 14).

Analysis of E2 Protein Gene Sequence

Virus samples collected at either day 2 or day 5 after infection of Verocells (infected at day 4 post-seeding) were selected to conduct ananalysis of genomic RNA sequence of the viral E2 structural protein.These samples are most representative for Vero cell confluence on rollerbottles. Tables 6 and 7 below summarize the percentage ofheterogeneities estimated for four amino acid (AA) positions based onthe nucleic acid sequence determined by Sanger sequencing. Table 6 showsdata for CHIKV-Δ5nsP3 grown at three different temperatures andharvested two days post-infection and Table 7 shows data forCHIKV-Δ5nsP3 grown at 28° C. and harvested 5 days post-infection.

TABLE 6 Analysis of RNA genome sequence for E2 viral protein from D2post-infection sample harvests. Shown are the estimated percentages ofnucleic acid heterogeneities corresponding to four AA positions(indicated in parentheses), as determined by Sanger sequencing. Theheterogeneity at nucleic acid position 9649 is a silent mutation. Timepost seeding in roller bottles D4 Temperature 28° C. 35° C. 37° C. MOI(TCID₅₀/cell) 0.1 0.01 0.001 0.1 0.01 0.001 0.1 0.01 0.001 Pos. 8543 0 00 0-5 0-5 0-5 0 0 0 (G55R) Pos. 8882 30 30 40-50 25 25 30- 25 25 30-(E168K) 50 50 Pos. 9119 0-5 0-5 0-5 5-10 10 10- 10- 10- 10- (E247K) 2020 20 20 Pos. 9649 0-5 5-10 10-20 5 5 5- 0-5 0-5 5-10 (A423, Silent) 10

TABLE 7 Analysis of RNA genome sequence for E2 viral protein from D5post-infection sample harvests. Shown are the estimated percentages ofnucleic acid heterogeneities corresponding to four AA positions(indicated in parentheses), as determined by Sanger sequencing. Theheterogeneity at nucleic acid position 9649 is a silent mutation. Timepost seeding in roller bottles D4 Temperature 28° C. MOI (TCID₅₀/cell)0.1 0.01 0.001 Pos. 8543 (G55R) 0 0  0 Pos. 8882 (E168K) 30-40 40-50 50Pos. 9119 (E247K) 0-5  0-10 10 Pos. 9649 (A423, Silent) 0-5 0-5 10-20

MOI, temperature, day of infection post-Vero cell seeding and day ofsample harvest all influenced the productivity and the quality ofCHIKV-Δ5nsP3 when produced in Vero cells. The strength of eachparameter, however, was of different importance. For example, theresults suggested a correlation between MOI and heterogeneity levels;i.e., the lower the viral input at infection, the higher the observedlevel of heterogeneity at harvest. The incubation temperature did notappear to impact the stability of the nucleotide sequence, with theexception of Pos. 9119 (E247K) where a higher level of heterogeneity wasobserved at 37° C. (Table 6). Also, the sample harvest collected laterin the viral kinetic triggered a slightly higher level of heterogeneityfor the same AA position.

To complete this first analysis, mathematical modelling of the raw datawas also performed (FIGS. 17 and 18). Only three nucleic acid positions(Pos. 8882, 9119, 9649) could be analyzed as no significant model wascalculated for the position 8543. As indicated in FIG. 17, temperatureand MOI had different impacts depending on the nucleic acid positionconsidered. The MOI significantly influenced the heterogeneitiesobserved for nucleic acid positions 8882 and 9649, but not for 9112. Thetemperature affected positions 9112 and 9649, but not 8882 (FIG. 17).MOI was shown to consistently influence the level of heterogeneity whenD2 and D5 post-infection harvest samples from 28° C.-produced virus werecompared-minimized levels of AA heterogeneity were observed when cellswere inoculated with a high quantity of virus (MOI 0.1 TCID₅₀/cell).This observation might prompt the establishment of new process parametersettings with a higher MOI as a means to achieve maximal virusproduction/titer stability while ensuring a low level of heterogeneitywithin the E2 protein. However, an MOI of 0.1 results in significantlyhigher consumption of GMP working virus seed bank, thus limiting itspractical industrial applicability.

Post-infection harvest day only impacted the variation of nucleic acidposition 9119 (FIG. 18).

SEQUENCES Nucleotide sequence of the CHIKV-Δ5nsP3 SEQ ID NO: 1GATGGCTGCGTGAGACACACGTAGCCTACCAGTTTCTTACTGCTCTACTCTGCAAAGCAAGAGATTAATAACCCATCATGGATCCTGTGTACGTGGACATAGACGCTGACAGCGCCTTTTTGAAGGCCCTGCAACGTGCGTACCCCATGTTTGAGGTGGAACCAAGGCAGGTCACACCGAATGACCATGCTAATGCTAGAGCGTTCTCGCATCTAGCTATAAAACTAATAGAGCAGGAAATTGACCCCGACTCAACCATCCTGGATATCGGCAGTGCGCCAGCAAGGAGGATGATGTCGGACAGGAAGTACCACTGCGTCTGCCCGATGCGCAGTGCGGAAGATCCCGAGAGACTCGCCAATTATGCGAGAAAGCTAGCATCTGCCGCAGGAAAAGTCCTGGACAGAAACATCTCTGGAAAGATCGGGGACTTACAAGCAGTAATGGCCGTGCCAGACACGGAGACGCCAACATTCTGCTTACACACAGACGTCTCATGTAGACAGAGAGCAGACGTCGCTATATACCAAGACGTCTATGCTGTACACGCACCCACGTCGCTATACCACCAGGCGATTAAAGGGGTCCGAGTGGCGTACTGGGTTGGGTTCGACACAACCCCGTTCATGTACAATGCCATGGCGGGTGCCTACCCCTCATACTCGACAAACTGGGCAGATGAGCAGGTACTGAAGGCTAAGAACATAGGATTATGTTCAACAGACCTGACGGAAGGTAGACGAGGCAAGTTGTCTATTATGAGAGGGAAAAAGCTAAAACCGTGCGACCGTGTGCTGTTCTCAGTAGGGTCAACGCTCTACCCGGAAAGCCGCAAGCTACTTAAGAGCTGGCACCTGCCATCGGTGTCCATTTAAAGGGCAAACTCAGCTTCACATGCCGCTGTGATACAGTGGTTTCGTGTGAGGGCTACGTCGTTAAGAGAATAACGATGAGCCCAGGCCTTTATGGAAAAACCACAGGGTATGCGGTAACCCACCACGCAGACGGATTCCTGATGTGCAAGACTACCGACACGGTTGACGGCGAAAGAATGTCATTCTCGGTGTGCACATACGTGCCGGCGACCATTTGTGATCAAATGACCGGCATCCTTGCTACAGAAGTCACGCCGGAGGATGCACAGAAGCTGTTGGTGGGGCTGAACCAGAGAATAGTGGTTAACGGCAGAACGCAACGGAATACGAACACCATGAAAAATTATCTGCTTCCCGTGGTCGCCCAAGCCTTCAGTAAGTGGGCAAAGGAGTGCCGGAAAGACATGGAAGATGAAAAACTCCTGGGGGTCAGAGAAAGAACACTGACCTGCTGCTGTCTATGGGCATTCAAGAAGCAGAAAACACACACGGTCTACAAGAGGCCTGATACCCAGTCAATTCAGAAGGTTCAGGCCGAGTTTGACAGCTTTGTGGTACCGAGTCTGTGGTCGTCCGGGTTGTCAATCCCTTTGAGGACTAGAATCAAATGGTTGTTAAGCAAGGTGCCAAAAACCGACCTGATCCCATACAGCGGAGACGCCCGAGAAGCCCGGGACGCAGAAAAAGAAGCAGAGGAAGAACGAGAAGCAGAACTGACTCGCGAAGCCCTACCACCTCTACAGGCAGCACAGGAAGATGTTCAGGTCGAAATCGACGTGGAACAGCTTGAGGACAGAGCGGGCGCAGGAATAATAGAGACTCCGAGAGGAGCTATCAAAGTTACTGCCCAACCAACAGACCACGTCGTGGGAGAGTACCTGGTACTCTCCCCGCAGACCGTACTACGTAGCCAGAAGCTCAGTCTGATTCACGCTTTGGCGGAGCAAGTGAAGACGTGCACGCACAACGGACGAGCAGGGAGGTATGCGGTCGAAGCGTACGACGGCCGAGTCCTAGTGCCCTCAGGCTATGCAATCTCGCCTGAAGACTTCCAGAGTCTAAGCGAAAGCGCAACGATGGTGTATAACGAAAGAGAGTTCGTAAACAGAAAGCTACACCATATTGCGATGCACGGACCAGCCCTGAACACCGACGAAGAGTCGTATGAGCTGGTGAGGGCAGAGAGGACAGAACACGAGTACGTCTACGACGTGGATCAGAGAAGATGCTGTAAGAAGGAAGAAGCCGCAGGACTGGTACTGGTGGGCGACTTGACTAATCCGCCCTACCACGAATTCGCATATGAAGGGCTAAAAATCCGCCCTGCCTGCCCATACAAAATTGCAGTCATAGGAGTCTTCGGAGTACCGGGATCTGGCAAGTCAGCTATTATCAAGAACCTAGTTACCAGGCAGGACCTGGTGACTAGCGGAAAGAAAGAAAACTGCCAAGAAATCACCACCGACGTGATGAGACAGAGAGGTCTAGAGATATCTGCACGTACGGTTGACTCGCTGCTCTTGAATGGATGCAACAGACCAGTCGACGTGTTGTACGTAGACGAGGCGTTTGCGTGCCACTCTGGAACGCTACTTGCTTTGATCGCCTTGGTGAGACCAAGGCAGAAAGTTGTACTTTGTGGTGACCCGAAGCAGTGCGGCTTCTTCAATATGATGCAGATGAAAGTCAACTATAATCACAACATCTGCACCCAAGTGTACCACAAAAGTATCTCCAGGCGGTGTACACTGCCTGTGACCGCCATTGTGTCATCGTTGCATTACGAAGGCAAAATGCGCACTACGAATGAGTACAACAAGCCGATTGTAGTGGACACTACAGGCTCAACAAAACCTGACCCTGGAGACCTCGTGTTAACGTGCTTCAGAGGGTGGGTTAAACAACTGCAAATTGACTATCGTGGATACGAGGTCATGACAGCAGCCGCATCCCAAGGGTTAACCAGAAAAGGAGTTTACGCAGTTAGACAAAAAGTTAATGAAAACCCGCTCTATGCATCAACGTCAGAGCACGTCAACGTACTCCTAACGCGTACGGAAGGTAAACTGGTATGGAAGACACTTTCCGGCGACCCGTGGATAAAGACGCTGCAGAACCCACCGAAAGGAAACTTCAAAGCAACTATTAAGGAGTGGGAGGTGGAGCATGCATCAATAATGGCGGGCATCTGCAGTCACCAAATGACCTTCGATACATTCCAAAATAAAGCCAACGTTTGTTGGGCTAAGAGCTTGGTCCCTATCCTCGAAACAGCGGGGATAAAACTAAATGATAGGCAGTGGTCTCAGATAATTCAAGCCTTCAAAGAAGACAAAGCATACTCACCTGAAGTAGCCCTGAATGAAATATGTACGCGCATGTATGGGGTGGATCTAGACAGCGGGCTATTTTCTAAACCGTTGGTGTCTGTGTATTACGCGGATAACCACTGGGATAATAGGCCTGGAGGGAAAATGTTCGGATTTAACCCCGAGGCAGCATCCATTCTAGAAAGAAAGTATCCATTCACAAAAGGGAAGTGGAACATCAACAAGCAGATCTGCGTGACTACCAGGAGGATAGAAGACTTTAACCCTACCACCAACATCATACCGGCCAACAGGAGACTACCACACTCATTAGTGGCCGAACACCGCCCAGTAAAAGGGGAAAGAATGGAATGGCTGGTTAACAAGATAAACGGCCACCACGTGCTCCTGGTCAGTGGCTATAACCTTGCACTGCCTACTAAGAGAGTCACTTGGGTAGCGCCGTTAGGTGTCCGCGGAGCGGACTACACATACAACCTAGAGTTGGGTCTGCCAGCAACGCTTGGTAGGTATGACCTAGTGGTCATAAACATCCACACACCTTTTCGCATACACCATTACCAACAGTGCGTCGACCACGCAATGAAACTGCAAATGCTCGGGGGTGACTCATTGAGACTGCTCAAACCGGGCGGCTCTCTATTGATCAGAGCATATGGTTACGCAGATAGAACCAGTGAACGAGTCATCTGCGTATTGGGACGCAAGTTTAGATCGTCTAGAGCGTTGAAACCACCATGTGTCACCAGCAACACTGAGATGTTTTTCCTATTCAGCAACTTTGACAATGGCAGAAGGAATTTCACAACTCATGTCATGAACAATCAACTGAATGCAGCCTTCGTAGGACAGGTCACCCGAGCAGGATGTGCACCGTCGTACCGGGTAAAACGCATGGACATCGCGAAGAACGATGAAGAGTGCGTAGTCAACGCCGCTAACCCTCGCGGGTTACCGGGTGGCGGTGTTTGCAAGGCAGTATACAAAAAATGGCCGGAGTCCTTTAAGAACAGTGCAACACCAGTGGGAACCGCAAAAACAGTTATGTGCGGTACGTATCCAGTAATCCACGCTGTTGGACCAAACTTCTCTAATTATTCGGAGTCTGAAGGGGACCGGGAATTGGCAGCTGCCTATCGAGAAGTCGCAAAGGAAGTAACTAGGCTGGGAGTAAATAGTGTAGCTATACCTCTCCTCTCCACAGGTGTATACTCAGGAGGGAAAGACAGGCTGACCCAGTCACTGAACCACCTCTTTACAGCCATGGACTCGACGGATGCAGACGTGGTCATCTACTGCCGCGACAAAGAATGGGAGAAGAAAATATCTGAGGCCATACAGATGCGGACCCAAGTAGAGCTGCTGGATGAGCACATCTCCATAGACTGCGATATTGTTCGCGTGCACCCTGACAGCAGCTTGGCAGGCAGAAAAGGATACAGCACCACGGAAGGCGCACTGTACTCATATCTAGAAGGGACCCGTTTTCATCAGACGGCTGTGGATATGGCGGAGATACATACTATGTGGCCAAAGCAAACAGAGGCCAATGAGCAAGTCTGCCTATATGCCCTGGGGGAAAGTATTGAATCGATCAGGCAGAAATGCCCGGTGGATGATGCAGACGCATCATCTCCCCCCAAAACTGTCCCGTGCCTTTGCCGTTACGCTATGACTCCAGAACGCGTCACCCGGCTTCGCATGAACCACGTCACAAGCATAATTGTGTGTTCTTCGTTTCCCCTCCCAAAGTACAAAATAGAAGGAGTGCAAAAAGTCAAATGCTCTAAGGTAATGCTATTTGACCACAACCTGCCATCGCGCGTAAGTCCAAGGGCTTATAGAGGTGCCGCTGCCGGTAACCTTGCGGCCGTGTCTGATTGGGTAATGAGCACCGTACCTGTCGCGCCGCCCAGAAGAAGGCGAGGGAGAAACCTGACTGTGACATGTGACGAGAGAGAAGGGAATATAACACCCATGGCTAGCGTCCGATTCTTTAGGGCAGAGCTGTGTCCGGTCGTACAAGAAACAGCGGAGACGCGTGACACAGCAATGTCTCTTCAGGCACCACCGAGTACCGCCACGGAACCGAATCATCCGCCGATCTCCTTCGGAGCATCAAGCGAGACGTTCCCCATTACATTTGGGGACTTCAACGAAGGAGAAATCGAAAGCTTGTCTTCTGAGCTACTAACTTTCGGAGACTTCTTACCAGGAGAAGTGGATGACTTGACAGACAGCGACTGGTCCACGTGCTCAGACACGGACGACGAGTTAAGACTAGACAGGGCAGGTGGGTATATATTCTCGTCGGACACCGGTCCAGGTCATTTACAACAGAAGTCAGTACGCCAGTCAGTGCTGCCGCTGAACACCCTGGAGGAACTCCACCAGGAGAAGTGTTACCCACCTAAGCTGGATGAAGCAAAGGAGCAACTATTACTTAAGAAACTCCAGGAGAGTGCATCCATGGCCAACAGAAGCAGGTATCAGTCGCGCAAAGTAGAAAACATGAAAGCAGCAATCATCCAGAGACTAAAGAGAGGCTGTAGACTATACTTAATGTCAGAGACCCCAAAAGTCCCTACTTACCGGACTACATATCCGGCGCCTGTGTACTCGCCTCCGATCAACGTCCGATTGTCCAATCCCGAGTCCGCAGTGGCAGCATGCAATGAGTTCTTAGCTAGAAACTATCCAACTGTCTCATCATACCAAATTACCGACGAGTATGATGCATATCTAGACATGGTGGACGGGTCGGAGAGTTGCCTGGACCGAGCGACATTCAATCCGTCAAAACTCAGGAGCTACCCGAAACAGCACGCTTACCACGCGCCCTCCATCAGAAGCGCTGTACCGTCCCCATTCCAGAACACACTACAGAATGTACTGGCAGCAGCCACGAAAAGAAACTGCAACGTCACACAGATGAGGGAATTACCCACTTTGGACTCAGCAGTATTCAACCTGGAGTGTTTCAAAAAATTCGCATGCAACCAAGAATACTGGGAAGAATTTGCTGCCAGCCCTATTAGGATAACAACTGAGAATTTAGCAACCTATGTTACTAAACTAAAAGGGCCAAAAGCAGCAGCGCTATTCGCAAAAACCCATAATCTACTGCCACTACAGGAAGTACCAATGGATAGGTTCACAGTAGATATGAAAAGGGACGTAAAGGTGACTCCTGGTACAAAGCATACAGAGGAAAGACCTAAGGTGCAGGTTATACAGGCGGCTGAACCCTTGGCGACAGCATACCTATGTGGGATTCACAGAGAGCTGGTTAGGAGGCTGAACGCCGTCCTCCTACCCAATGTACATACACTATTTGACATGTCTGCCGAGGATTTCGATGCCATCATAGCCGCACACTTTAAGCCAGGAGACACTGTTTTGGAAACGGACATAGCCTCCTTTGATAAGAGCCAAGATGATTCACTTGCGCTTACTGCTTTGATGCTGTTAGAGGATTTAGGGGTGGATCACTCCCTGCTCGACTTGATAGAGGCTGCTTTCCGAGAGATTTCCAGCTGTCACCTACCGACAGGTACGCGCTTCAAGTTCGGCGCCATGATGAAATCAGGTATGTTCCTAACTCTGTTCGTCAACACATTGTTAAACATCACCATCGCCAGCCGAGTGCTGGAAGATCGTCTGACAAAATCCGCGTGCGCGGCCTTCATCGGCGACGACAACATAATACATGGAGTCGTCTCCGATGAATTGATGGCAGCCAGATGTGCCACTTGGATGAACATGGAAGTGAAGATCATAGATGCAGTTGTATCCTTGAAAGCCCCTTACTTTTGTGGAGGGTTTATACTGCACGATACTGTGACAGGAACAGCTTCCAGAGTGGCAGACCCGCTAAAAAGGCTTTTTAAACTGGGCAAACCGCTAGCGGCAGGTGACGAACAAGATGAAGATAGAAGACGAGCGCTGGCTGACGAAGTGATCAGATGGCAACGAACAGGGCTAATTGATGAGCTGGAGAAAGCGGTATACTCTAGGTACGAAGTGCAGGGTATATCAGTTGTGGTAATGTCCATGGCCACCTTTGCAAGCTCCAGATCCAACTTCGAGAAGCTCAGAGGACCCGTCATAACTTTGTACGGCGGTCCTAAATAGGTACGCACTACAGCTACCTATTTTGCAGAAGCCGACAGCAAGTATCTAAACACTAATCAGCTACAATGGAGTTCATCCCAACCCAAACTTTTTACAATAGGAGGTACCAGCCTCGACCCTGGACTCCGCGCCCTACTATCCAAGTCATCAGGCCCAGACCGCGCCCTCAGAGGCAAGCTGGGCAACTTGCCCAGCTGATCTCAGCAGTTAATAAACTGACAATGCGCGCGGTACCACAACAGAAGCCACGCAGGAATCGGAAGAATAAGAAGCAAAAGCAAAAACAACAGGCGCCACAAAACAACACAAATCAAAAGAAGCAGCCACCTAAAAAGAAACCGGCTCAAAAGAAAAAGAAGCCGGGCCGCAGAGAGAGGATGTGCATGAAAATCGAAAATGATTGTATTTTCGAAGTCAAGCACGAAGGTAAGGTAACAGGTTACGCGTGCCTGGTGGGGGACAAAGTAATGAAACCAGCACACGTAAAGGGGACCATCGATAACGCGGACCTGGCCAAACTGGCCTTTAAGCGGTCATCTAAGTATGACCTTGAATGCGCCCAGATACCCGTGCACATGAAGTCCGACGCTTCGAAGTTCACCCATGAGAAACCGGAGGGGTACTACAACTGGCACCACGGAGCAGTACAGTACTCAGGAGGCCGGTTCACCATCCCTACAGGTGCTGGCAAACCAGGGGACAGCGGCAGACCGATCTTCGACAACAAGGGACGCGTGGTGGCCATAGTCTTAGGAGGAGCTAATGAAGGAGCCCGTACAGCCCTCTCGGTGGTGACCTGGAATAAAGACATTGTCACTAAAATCACCCCCGAGGGGGCCGAAGAGTGGAGTCTTGCCATCCCAGTTATGTGCCTGTTTGGCAAACACCACGTTCCCCTGCTCCCAGCCCCCTTGCACGCCCTGCTGCTACGAAAAGGAACCGGAGGAAACCCTACGCATGCTTGAGGACAACGTCATGAGACCTGGGTACTATCAGCTGCTACAAGCATCCTTAACATGTTCTCCCCACCGCCAGCGACGCAGCACCAAGGACAACTTCAATGTCTATAAAGCCACAAGACCATACTTAGCTCACTGTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCACTAGAACGCATCAGAAATGAAGCGACAGACGGGACGCTGAAAATCCAGGTCTCCTTGCAAATCGGAATAAAGACGGATGACAGCCACGATTGGACCAAGCTGCGTTATATGGACAACCACATGCCAGCAGACGCAGAGAGGGCGGGGCTATTTGTAAGAACATCAGCACCGTGTACGATTACTGGAACAATGGGACACTTCATCCTGGCCCGATGTCCAAAAGGGGAAACTCTGACGGTGGGATTCACTGACAGTAGGAAGATTAGTCACTCATGTACGCACCCATTTCACCACGACCCTCCTGTGATAGGTCGGGAAAAATTCCATTCCCGACCGCAGCACGGTAAAGAGCTACCTTGCAGCACGTACGTGCAGAGCACCGCCGCAACTACCGAGGAGATAGAGGTACACATGCCCCCAGACACCCCTGATCGCACATTAATGTCACAACAGTCCGGCAACGTAAAGATCACAGTCAATGGCCAGACGGTGCGGTACAAGTGTAATTGCGGTGGCTCAAATGAAGGACTAACAACTACAGACAAAGTGATTAATAACTGCAAGGTTGATCAATGTCATGCCGCGGTCACCAATCACAAAAAGTGGCAGTATAACTCCCCTCTGGTCCCGCGTAATGCTGAACTTGGGGACCGAAAAGGAAAAATTCACATCCCGTTTCCGCTGGCAAATGTAACATGCAGGGTGCCTAAAGCAAGGAACCCCACCGTGACGTACGGGAAAAACCAAGTCATCATGCTACTGTATCCTGACCACCCAACACTCCTGTCCTACCGGAATATGGGAGAAGAACCAAACTATCAAGAAGAGTGGGTGATGCATAAGAAGGAAGTCGTGCTAACCGTGCCGACTGAAGGGCTCGAGGTCACGTGGGGCAACAACGAGCCGTATAAGTATTGGCCGCAGTTATCTACAAACGGTACAGCCCATGGCCACCCGCATGAGATAATTCTGTATTATTATGAGCTGTACCCCACTATGACTGTAGTAGTTGTGTCAGTGGCCACGTTCATACTCCTGTCGATGGTGGGTATGGCAGCGGGGATGTGCATGTGTGCACGACGCAGATGCATCACACCGTATGAACTGACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATCAGAACAGCTAAAGCGGCCACATACCAAGAGGCTGCGATATACCTGTGGAACGAGCAGCAACCTTTGTTTTGGCTACAAGCCCTTATTCCGCTGGCAGCCCTGATTGTTCTATGCAACTGTCTGAGACTCTTACCATGCTGCTGTAAAACGTTGGCTTTTTTAGCCGTAATGAGCGTCGGTGCCCACACTGTGAGCGCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAGTCAATAGACCTGGCTACAGCCCCATGGTATTGGAGATGGAACTACTGTCAGTCACTTTGGAGCCAACACTATCGCTTGATTACATCACGTGCGAGTACAAAACCGTCATCCCGTCTCCGTACGTGAAGTGCTGCGGTACAGCAGAGTGCAAGGACAAAAACCTACCTGACTACAGCTGTAAGGTCTTCACCGGCGTCTACCCATTTATGTGGGGCGGCGCCTACTGCTTCTGCGACGCTGAAAACACGCAGTTGAGCGAAGCACACGTGGAGAAGTCCGAATCATGCAAAACAGAATTTGCATCAGCATACAGGGCTCATACCGCATCTGCATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAACATCACTGTAACTGCCTATGCAAACGGCGACCATGCCGTCACAGTTAAGGACGCCAAATTCATTGTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAACAAAATTGTGGTGTACAAAGGTGACGTCTATAACATGGACTACCCGCCCTTTGGCGCAGGAAGACCAGGACAATTTGGCGATATCCAAAGTCGCACACCTGAGAGTAAAGACGTCTATGCTAATACACAACTGGTACTGCAGAGACCGGCTGTGGGTACGGTACACGTGCCATACTCTCAGGCACCATCTGGCTTTAAGTATTGGCTAAAAGAACGCGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGCCAAATAGCAACAAACCCGGTAAGAGCGGTGAACTGCGCCGTAGGGAACATGCCCATCTCCATCGACATACCGGAAGCGGCCTTCACTAGGGTCGTCGACGCGCCCTCTTTAACGGACATGTCGTGCGAGGTACCAGCCTGCACCCATTCCTCAGACTTTGGGGGCGTCGCCATTATTAAATATGCAGCCAGCAAGAAAGGCAAGTGTGCGGTGCATTCGATGACTAACGCCGTCACTATTCGGGAAGCTGAGATAGAAGTTGAAGGGAATTCTCAGCTGCAAATCTCTTTCTCGACGGCCTTAGCCAGCGCCGAATTCCGCGTACAAGTCTGTTCTACACAAGTACACTGTGCAGCCGAGTGCCACCCCCCGAAGGACCACATAGTCAACTACCCGGCGTCACATACCACCCTCGGGGTCCAGGACATCTCCGCTACGGCGATGTCATGGGTGCAGAAGATCACGGGAGGTGTGGGACTGGTTGTTGCTGTTGCCGCACTGATTCTAATCGTGGTGCTATGCGTGTCGTTCAGCAGGCACTAACTTGACAATTAAGTATGAAGGTATATGTGTCCCCTAAGAGACACACTGTACATAGCAAATAATCTATAGATCAAAGGGCTACGCAACCCCTGAATAGTAACAAAATACAAAATCACTAAAAATTATAAAAACAGAAAAATACATAAATAGGTATACGTGTCCCCTAAGAGACACATTGTATGTAGGTGATAAGTATAGATCAAAGGGCCGAATAACCCCTGAATAGTAACAAAATATGAAAATCAATAAAAATCATAAAATAGAAAAACCATAAACAGAAGTAGTTCAAAGGGCTATAAAACCCCTGAATAGTAACAAAACATAAAATTAATAAAAATCAAATGAATACCATAATTGGCAAACGGAAGAGATGTAGGTACTTAAGCTTCCTAAAAGCAGCCGAACTCACTTTGAGAAGTAGGCATAGCATACCGAACTCTTCCACGATTCTCCGAACCCACAGGGACGTAGGAGATGTTATTTTGTTTTTAATATTTCAAAAAAAAAAAAAAAAAAAAAAAAAmino acid sequence of E2 protein from LR22006_OPY1 Chikungunya virus strain - aminoacids 339-742 from structural polyprotein GenBank Accession: ABD95938.1 (1-1248 aa)SEQ ID NO: 2STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKASome E2 variants identified hereinE168K variant of E2 protein from Chikungunya virus SEQ ID NO: 3STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIK VHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAG55R variant of E2 protein from Chikungunya virus SEQ ID NO: 4STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQI RIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAE247K variant of E2 protein from Chikungunya virus SEQ ID NO: 5STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAK LGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAG82R variant of E2 protein from Chikungunya virus SEQ ID NO: 6STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAR LFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFFISRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEWLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAH232Y variant of E2 protein from Chikungunya virus SEQ ID NO: 7STKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHSCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDHTLMSQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKVDQCHAAVTN YKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVMHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVATFILLSMVGMAAGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAPrimer 1F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 8TTAGGATCCGATGGCTGCGTGAGACAC Primer 1R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 9 TAACTCGAGCCGTCAGGTCTGTTGAACATPrimer 2F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 10TTAGGATCCTACCACCAGGCGATTAAAG Primer 2R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 11 TAACTCGAGCTTTGCCCACTTACTGAAGGPrimer 3F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 12TTAGGATCCTGCTACAGAAGTCACGCC Primer 3R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 13 TAACTCGAGGCCAAAGCGTGAATCAGPrimer 4F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 14TTAGGATCCAACAGCTTGAGGACAGAGCG Primer 4R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 15 TAACTCGAGCTCTGTCTCATCACGTCGGPrimer SF for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 16TTAGGATCCAAATTGCAGTCATAGGAGTCTTC Primer 5R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 17 TAACTCGAGAGTACGTTGACGTGCTCTGAPrimer 6F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 18TTAGGATCCGTGGGTTAAACAACTGCAAA Primer 6R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 19 TAACTCGAGGGTTAAAGTCTTCTATCCTCCTGGPrimer 7F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 20TTAGGATCCGGATAACCACTGGGATAATAGG Primer 7R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 21 TAACTCGAGAGTTGTGAAATTCCTTCTGCCPrimer 8F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 22TTAGGATCCCGCAGATAGAACCAGTGAAC Primer 8R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 23 TAACTCGAGCAGCAGCTCTACTTGGGTCPrimer 9F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 24TTAGGATCCAGGAGGGAAAGACAGGCT Primer 9R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 25 TAACTCGAGCCCTCGCCTTCTTCTGPrimer 10F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 26TTAGGATCCCAAAATAGAAGGAGTGCAAAAAG Primer l0R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 27 TAACTCGAGCCTGAGTTTCTTAAGTAATAGTTGCPrimer 11F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 28TTAGGATCCACCGGTCCAGGTCATTTA Primer 11R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 29 TAACTCGAGGCAGCAAATTCTTCCCAGPrimer 12F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 30TTAGGATCCCCATTCCAGAACACACTACAG Primer 12R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 31 TAACTCGAGATACCTGATTTCATCATGGCPrimer 13F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 32TTAGGATCCCCTTTGATAAGAGCCAAGATG Primer 13R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 33 TAACTCGAGTACAAAGTTATGACGGGTCCTPrimer 14F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 34TTAGGATCCCAACGAACAGGGCTAATTG Primer 14R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 35 TAACTCGAGGACCGCTTAAAGGCCAGPrimer 15F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 36TTAGGATCCGTGCATGAAAATCGAAANTG Primer 15R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 37 TAACTCGAGTGGTCTTGTGGCTTTATAGACAPrimer 16F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 38TTAGGATCCAACCGGAGGAAACCCTAC Primer 16R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 39 TAACTCGAGGTACCGCACCGTCTGGPrimer 17F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 40TTAGGATCCAGCTACCTTGCAGCACGT Primer 17R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 41 TAACTCGAGCCCACCATCGACAGGPrimer 18F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 42TTAGGATCCCGAGCCGTATAAGTATTGGC Primer 18R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 43 TAACTCGAGCGCCGGTGAAGACCTTACPrimer 19F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 44TTAGGATCCACTACTGTCAGTCACTTTGGAGC Primer 19R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 45 TAACTCGAGTACCGGGTTTGTTGCTATTTPrimer 20F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 46TTAGGATCCCACAACTGGTACTGCAGAGAC Primer 20R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 47 TAACTCGAGGCGTAGCCCTTTGATCTATAGPrimer 21F for CHIKV-Δ5nsP3 sequencing SEQ ID NO: 48TTAGGATCCGGTGCTATGCGTGTCGT Primer 21R for CHIKV-Δ5nsP3 sequencingSEQ ID NO: 49 TAACTCGAGATCTCCTACGTCCCTGTGGG

1. A pharmaceutical composition comprising: (i) CHIKV-Δ5nsP3 particlesexpressing an E2 structural protein as defined by the amino acidsequence of SEQ ID NO: 2; (ii) CHIKV-Δ5nsP3 particles expressing an E2structural protein having a mutation E168K in the amino acid sequence ofSEQ ID NO: 2; and (iii) optionally a pharmaceutically acceptableexcipient, wherein 1-50% of the CHIKV-Δ5nsP3 particles present in thecomposition express the E2 structural protein having the E168K mutation.2. The pharmaceutical composition according to claim 1, wherein saidcomposition induces neutralizing antibodies against CHIKV-Δ5nsP3 in amouse immunized with said pharmaceutical composition resulting in aserum comprising said neutralizing antibodies, and wherein said serumneutralizes Chikungunya virus (CHIKV) infection of Vero cells at least50% in an in vitro neutralization assay at a 1:80 serum dilution.
 3. Thepharmaceutical composition according to claim 1, wherein the compositioninduces neutralizing antibodies against CHIKV-Δ5nsP3 in a mouseimmunized with said pharmaceutical composition resulting in a serumcomprising said neutralizing antibodies, and wherein said serumneutralizes Chikungunya virus (CHIKV) infection of Vero cells at least80% in an in vitro neutralization assay at a 1:80 serum dilution.
 4. Thepharmaceutical composition according to claim 1, wherein the compositioninduces neutralizing antibodies against CHIKV-Δ5nsP3 in a mouseimmunized with said pharmaceutical composition resulting in a serumcomprising said neutralizing antibodies, and wherein said serumneutralizes Chikungunya virus (CHIKV) infection of Vero cells at least90% in an in vitro neutralization assay at a 1:80 serum dilution.
 5. Thepharmaceutical composition according to claim 1, wherein the compositioninduces neutralizing antibodies against CHIKV-Δ5nsP3 in a mouseimmunized with said pharmaceutical composition resulting in a serumcomprising said neutralizing antibodies, and wherein said serumneutralizes Chikungunya virus (CHIKV) infection of Vero cells at least95% in an in vitro neutralization assay at a 1:80 serum dilution.
 6. Thepharmaceutical composition according to claim 1, wherein saidCHIKV-Δ5nsP3 particles in (i) are defined by the polynucleotide sequenceof SEQ ID NO:
 1. 7. The pharmaceutical composition according to claim 1,wherein said CHIKV-Δ5nsP3 particles in (ii) further comprise a G55Rmutation in the E2 structural protein.
 8. The pharmaceutical compositionaccording to claim 1, wherein said pharmaceutical composition comprisesat least 103, at least 10⁴ CHIKV-Δ5nsP3 particles that express an E2structural protein as defined by the amino acid sequence of SEQ ID NO:2.
 9. The pharmaceutical composition according to claim 1, wherein saidpharmaceutical composition comprises about 10⁴ CHIKV-Δ5nsP3 particlesthat express an E2 structural protein as defined by the amino acidsequence of SEQ ID NO:
 2. 10. The pharmaceutical composition accordingto claim 1, wherein said pharmaceutical composition is a one-shotpharmaceutical composition.
 11. The pharmaceutical composition accordingto claim 1, wherein said pharmaceutical composition is a vaccine. 12.The pharmaceutical composition according to claim 11, wherein saidpharmaceutical composition is provided in a lyophilized form.
 13. Aprocess for producing a pharmaceutical composition comprisingCHIKV-Δ5nsP3 particles which express an E2 structural protein as definedby the amino acid sequence of SEQ ID NO: 2 comprising the step of:growing CHIKV-Δ5nsP3 particles on host cells in such a way as tominimize the presence of immunogenicity-reducing mutations of the virus.14. The process according to claim 13, wherein said CHIKV-Δ5nsP3particles are defined by a polynucleotide sequence of SEQ ID NO: 1 andare passaged on host cells in culture less than five times.
 15. Theprocess according to claim 14, wherein said CHIKV-Δ5nsP3 particles aredefined by a polynucleotide sequence of SEQ ID NO: 1 and are passaged onhost cells in culture at most three times.
 16. A method of protectionagainst disease caused by Chikungunya virus in a subject in needthereof, comprising administering to the subject an effective amount ofthe pharmaceutical composition according to claim
 1. 17. The methodaccording to claim 16, wherein said pharmaceutical composition comprisesan effective amount of CHIKV-Δ5nsP3 particles which express an E2structural protein as defined by the amino acid sequence of SEQ ID NO:2, wherein said effective amount is defined as an amount sufficient toprevent Chikungunya virus viremia in a vaccinated subject.
 18. Themethod according to claim 16, wherein the subject is a human subject.